Carboxylalkyl peptide derivatives

ABSTRACT

This invention encompasses novel carboxyalkyl peptide derivatives which are collagenase inhibitors.

BACKGROUND OF THE INVENTION

This application is a continuation-in-part application of priorcopending application Ser. No. 599307, filed Apr. 12, 1984, now U.S.Pat. No. 4,511,504.

This invention relates to novel compounds having pharmacologicalactivity, to the production thereof, to compositions containing them,and to their use in the treatment or management of conditions ordiseases, e.g., rheumatoid arthritis, in which collagenase promotedcollagen breakdown is a causative factor.

We have now found a group of compounds which act as inhibitors ofmammalian collagenase which initiates collagen breakdown. Extensiveproteolytic enzyme promoted degradation of articular cartilage isassociated with joint destruction in rheumatoid arthritis. Collagen isone of the major components of the protein matrix of joint cartilage andbone. Histological observations of rheumatoid lesions have establishedthat such lesions are characterized by the proliferation of synoviallining cells with subsequent neovascularization and infiltration bylymphocytes, macrophages and plasma cells, collectively referred to assoft tissue or "pannus". The importance of such soft tissue in cartilageerosion has been well demonstrated.

Evanson and coworkers, for example, found that large amounts of neutralcollagenase are produced by pannus tissue [Evanson, J. M., et al.,Arthritis & Rheum., 27, 2639-2651, 1968]. More recently, others haveconfirmed that neutral collagenase plays an important degradative rolein the arthritic joints of experimental animals [see Cambray, et al.,Rheumatol Int. 1, 11-16 and 17-20, 1981] and in humans [Cawston, et al.,Arthritis & Rheum., 27, 285-290, 1984].

A mono-specific antiserum to purified synovial collagenase has been usedto localize the enzyme in rheumatoid tissues [Wooley, et al., Eur. J.Biochem., 69, 421-428, 1976]. Immunoreactive collagenase was detected inhigh density at the cartilage-pannus junction--the site of jointerosion--but not in the cartilage matrix or synovial tissue remote fromthe cartilage-pannus junction [Wooley, et al., Arthritis & Rheumatism,20, 1231-1349.] Wooley, et al., (Science, 200, 773-775, 1978) havefurther identified a sub-population of synovial cells responsible forcollagenase production.

Thus, the foregoing observations have provided conclusive evidence thatcollagenase is directly involved in the cartilage erosion process seenin rheumatoid arthritis. Accordingly, the compounds of the presentinvention which specifically inhibit mammalian collagenase arepharmacologically useful in the treatment of rheumatoid arthritis andrelated diseases in which collagenolytic activity is a contributingfactor, such as corneal ulceration, peridontal disease, tumor invasion,dystrophic epidermolysis bullosa, etc.

These compounds have substantially no angiotensin converting enzyme(ACE)-inhibiting activity. ACE inhibitors are described in EuropeanAppl. No. A-0012401. ACE is a carboxydipeptidase--it cleaves a peptidesubstrate two residues from the C-terminus. Consequently the C-terminalcarboxylic acid is a prime recognition site for both substrates andinhibitors; removal of this ionic binding group drastically reducesinhibitory potency. Collagenase, on the other hand, is an endopeptidaseand, as such, has no prerequisite for this binding interaction.Additionally, the structure of collagen differs essentially fromangiotensin-I which is a decapeptide and is cleaved at aphenylalanine-histidine bond to give an octapeptide (angiotensin-II) anda dipeptide (histidylleucine). Collagen is much more complex, in being atriple helix, each strand of the helix containing of the order of 1,000amino acid residues, the sequence of amino acids around the site cleavedby collagenase being completely different from that around the cleavagesite of Angiotensin I. Collagenase cleaves this triple helix at a singlelocus on each chain approximately two-thirds of the way along the chainfrom the N-terminus. The amide bond which is cleaved by collagenase iseither a glycine-leucine or a glycine-isoleucine bond.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides compounds of the general formula I:##STR1## and pharmaceutically acceptable salts thereof in which

n=1-4

R¹ represents hydroxy, alkoxy, aralkoxy or hydroxy-amino;

R² represents hydrogen or alkyl;

R³ represents

hydrogen,

alkyl,

substituted alkyl wherein the substituent may be one or more of thegroups selected from hydroxy, alkoxy, aryloxy, aralkoxy, mercapto,alkylthio, arylthio, alkylsulphinyl (e.g. SOCH₃), alkylsulphonyl (e.g.SO₂ CH₃), carboxy, carboxamido (e.g. CONH₂), carboxyalkyl (e.g. CO₂CH₃), carboxyaralkyl (e.g. CO₂ CH₂ Ph), aralkoxycarbonylamino (e.g.NHCOOCH₂ Ph), amino, dialkylamino, acylamino (e.g. NHCOCH₃), aroylamino(e.g. NHCOPh) and trihalomethyl (e.g. CF₃),

aralkyl,

substituted aralkyl wherein the substituent on the aryl moiety may beone or more groups selected from halogen (e.g. fluorine, chlorine,bromine, iodine), alkyl, hydroxy, alkoxy, aralkoxy, amino, aminomethyl(CH₂ NH₂), cyano, alkylamino, dialkylamino, carboxy, sulphonamido,alkylthio, nitro and phenyl,

or heteroaralkyl;

Y represents NR⁴ wherein R⁴ represents H or alkyl; or for certain valuesof A¹, A² may alternatively be a direct chemical bond.

When Y represents NR⁴,

A¹ represents a group of formula R⁵ wherein

R⁵ may be

hydrogen,

alkyl,

aralkyl,

aryl,

substituted aryl wherein the substituent may be one or more groupsselected from halogen alkyl, hydroxy, alkoxy, aralkoxy, aralkoxyamino,aminomethyl, cyano, acylamino, dialkylamino, carboxy, sulphonamido,alkylthio, nitro and phenyl,

acyl (e.g. CH₃ CO),

aroyl (e.g. PhCO),

aralkylacyl (e.g. PhCH₂ CO),

alkoxycarbonyl (e.g. (CH₃)₃ OCO),

or aralkoxycarbonyl (e.g. PhCH₂ OCO);

A¹ may also represent a group of the formula: ##STR2## wherein

R⁶ represents a group having the meanings defined above for R⁵ ;

R⁷ and R⁸ which may be the same or different represent hydrogen, alkylor aralkyl; or

R⁷ and R⁸ may together represent an alkylene chain of 2-4 carbon atomsso to form with the adjacent nitrogen atom a nitrogen-containing ringhaving 4-6 atoms;

R⁹ represents

hydrogen,

alkyl,

substituted alkyl wherein the substituent is exactly as defined for thismoiety above,

aralkyl,

substituted aralkyl wherein the substituent is exactly as defined forthis moiety above,

or heteroaralkyl;

A² represents a group of the formula ##STR3## wherein

R¹⁰ and R¹¹ which may be the same or different represent groups havingthe meanings given above for R⁷ or together represent an alkylene chainof 2-4 carbon atoms so as to form with the adjacent nitrogen anitrogen-containing ring having 4 to 6 atoms;

R¹² represents a group having the meanings given above for R⁹.

Additionally, A¹ and A² taken together may represent

hydrogen,

alkyl,

aralkyl,

heteroaralkyl,

alkylsulphonyl,

arylsulphonyl,

aralkylsulphonyl,

or a group R¹³ CO wherein R¹³ represents

hydrogen,

alkyl,

alkoxy,

aryl,

aralkyl,

aralkoxy,

substituted aryl (as defined in R³), substituted aralkyl (as defined inR³) and substituted aralkoxy wherein the substituent on the aromaticmoiety of the aralkoxy is as defined for aralkyl

phenethenyl (PhCH═CH--),

phenethynyl (PhC.tbd.C--),

alkylamino,

arylamino,

aralkylamino,

or dialkylamino;

In a further aspect of this invention, Y may also represent a directchemical bond. In this instance, A¹ and A² taken together represent

hydrogen,

alkyl,

aryl,

alkoxy,

aralkoxy,

substituted aryl (as in R³) and substituted aralkoxy (as in R³) whereinthe substituent on the aromatic moiety of the aralkoxy is as defined foraralkyl,

hydroxy,

mercapto,

alkylthio,

arylthio,

aralkylthio,

carboxy,

or carboxyalkyl;

A³ represents a group of the formula

R¹⁴

or ##STR4## wherein

R¹⁴ represents amino,

alkylamine,

dialkylamino,

hydroxyamino,

or aralkylamino,

and R¹⁵, R¹⁶ and R¹⁷ which may be the same or different represent groupshaving the meaning given above for R¹⁰, R¹¹ and R¹² respectively and

R¹⁸ represents

amino,

alkylamino,

dialkylamino,

substituted alkylamino wherein the substituent is amino, hydroxy,alkoxy, carboxy, carboxamido, carboxyalkyl, alkylthio, alkylsulphinyl oralkylsulphonyl,

hydroxyamino,

alkoxyamino,

aralkylamino,

alkoxy,

aralkoxy,

or alkylaminoalkoxy.

all with the exception that when A³ is alkylamino one of R² and R³ isnot hydrogen and the other alkyl or hydroxyalkyl.

DETAILED DESCRIPTION OF THE INVENTION

The term alkyl as used herein to designate a group or a part thereofincludes reference to both straight and branched alkyl groups and tocycloalkyl groups which may contain from 1 to 10, preferably 1 to 6,carbon atoms in the case of straight or branched chain non-cyclic alkylgroups (for example methyl, ethyl, propyl, isopropyl) and from 3 to 10,preferably 3 to 7 in the case of cyclic alkyl groups (for examplecyclopentyl, norbornyl).

By the term aryl, is meant phenyl or naphthyl.

The terms aralkyl and aralkoxy include in particular those groupscontaining 1 to 4 carbon atoms in the alkyl portion, and those groups inwhich aryl has the meaning just given.

By the term heteroaralkyl we mean in particular groups containing 1 to 4carbon atoms in the alkyl moiety. The term heteroaryl includes forexample, pyridyl, thienyl, furyl, indolyl, imidazolyl and thiazolyl.

Typical pharmaceutically acceptable addition salts are those derivedfrom mineral and organic acids such as hydrochloric, hydrobromic,hydroiodic, p-toluene sulphonic, sulphuric, perchloric, acetic, benzoic,trifluoroacetic and the like.

There are several chiral centres in the compounds according to theinvention because of the presence of asymmetric carbon atoms. Thesecentres may be racemised or in any optically active form. We have foundsurprisingly that those compounds in which the chiral centre indicatedbelow by an asterisk in the group shown is in the R form are preferred.##STR5##

Certain groups of compounds according to the invention are preferred,these including the following. A group of preferred compounds are thosein which the group A³ has the following meaning ##STR6## in which R¹⁷represents substituted alkyl (wherein the substituent is alkoxy,aralkoxy, alkoxycarbonylamino, aralkoxycarbonylamino, carboxyalkyl orcarboxyaralkyl); or substituted aralkyl (wherein the aryl substituent isone or more groups selected from alkyl, alkoxy, alkyl thio or aralkoxy).In this preferred group of compounds, R³ should have the meaningsdescribed hereinafter but excluding aralkyl or heteroalkyl. Within thisdefinition of A³, there is a preferred subclass of compounds in which A¹+A² taken together represent H, Y is a direct chemical bond, R²represents H, and R³ represents alkyl or substituted alkyl where thesubstituent(s) is one or more trifluoromethyl groups. Therefore thisfirst sub-class of preferred compounds may be defined by the formula##STR7## wherein R³ and R¹⁷ are as defined above. A most preferred setof compounds within this group are those in which R¹⁷ is benzyloxymethyl(PhCH₂ OCH₂ --), 1-benzyloxyethyl (PhCH₂ OCH(CH₃)--),4-benzyloxyphenylmethyl (4--PhCH₂ OC₆ H₄ CH₂ --) or4-methoxyphenylmethyl (4--CH₃ OC₆ H₄ CH₂ --).

In a second preferred sub-class of compounds within the preferreddefinition of A³, Y represents NR⁴, and A¹ +A² represent a group R¹³ COwherein R¹³ represents

alkyl,

aryl,

aralkyl,

aralkoxy,

substituted aryl, substituted aralkyl and substituted aralkoxy whereinthe substituent on the aromatic moiety is exactly as definedhereinbefore,

alkylamino,

arylamino,

aralkylamino

or dialkylamino.

Therefore this second sub-class of preferred compounds may be defined bythe formula ##STR8##

Particularly preferred examples are those in which R⁴ is H and R³, R¹⁷and R¹⁸ are as defined for the first preferred sub-class of compounds. Amost preferred series of compounds within this sub-class is where n is2, R¹³ is benzyloxy (PhCH₂ O), substituted benzyloxy (where the aromaticsubstituent is selected from 4-chloro, 2-chloro, 4-methyl, 4-nitro or4-amino), benzylamino (PhCH₂ NH), phenyl or substituted phenyl (wherethe aromatic substituent is selected from 4-chloro, 2-chloro, 4-methyl,4-nitro or 4-amino).

A further preferred embodiment of the invention is a compound of theformula ##STR9## and the pharmaceutically acceptable acid addition saltsthereof wherein x represents hydrogen, alkoxy or benzyloxy; y representsa radical selected from alkyl, alkylthioalkyl, ##STR10## wherein v is 2or 3, ##STR11## wherein z represents hydrogen or nitro; W₁ and W₂represent methyl or trifluoromethyl; and R¹ represents hydroxy or alkoxyand the stereochemistry of the carbon marked by the asterisk is R.

A still further especially preferred embodiment of the invention arecompounds of the formula: ##STR12## and the pharmaceutically acceptableacid addition salts thereof wherein R'₂ represents hydroxy, alkoxy,cycloalkoxy, aralkoxy or substituted alkoxy wherein the substituent isselected from alkylaminocarbonyl or the group ##STR13## R'₃ representsalkylamino or aralkylamino and the stereochemistry of the carbon atommarked by the asterisk is R, S or a diastereomeric mixture thereof withR configuration being preferred.

The compounds according to the invention exhibit inhibitory actionagainst collagenase. This was determined following the procedure ofCawston and Barrett, Anal. Biochem., 99, 340-345 (1979) whereby the 1 mMof the inhibitor being tested or dilutions thereof are incubated at 37°C. for 16 hours with native collagen and collagenase (buffered with TrisHCl--CaCl₂ ; pH 7.6). The collagen is acetyl ¹⁴ C collagen. The samplesare centrifuged to sediment undigested collagen and an aliquot of theradiactive supernatant removed for assay on a scintillation counter as ameasure of hydrolysis. The collagenase activity in the presence of 1 mMinhibitor, or a dilution thereof, is compared to activity in a controldevoid of inhibitor and the results reported as that inhibitorconcentration effecting 50% inhibition of the collagenase. Table IIillustrates the activity of compounds of this invention.

For use in treatment of rheumatoid arthritis the compounds of thisinvention can be administered by any convenient route preferably in theform of a pharmaceutical composition adapted to such route and in a doseeffective for the intended treatment. In the treatment of arthritisadministration may conveniently be by the oral route or by injectionintraarticularly into the affected joint. The daily dosage for a 70kilogram mammal will be in the range of 10 milligrams to 1 gram.

The compounds of this invention can be formulated in compositions suchas tablets, capsules or elixirs for oral administration or in sterilesolutions or suspensions for parenteral administration. About 10 to 500mg of a compound according to the invention is compounded with aphysiologically acceptable vehicle, carrier, excipient, binder,preservative, stabilizer, flavour, etc., in a unit dosage from as calledfor by accepted pharmaceutical practice. (See for example, Remington'sPharmaceutical Science Mach Publishing Co., Easton, Penn. 1965). Theamount of active substance in these compositions or preparations is suchthat a suitable dosage in the range indicated is obtained.

The compounds according to the invention may be made by methods whichare generally known in peptide chemistry for analogous compounds. Inparticular it is to be understood that reactive groups not involved in aparticular reaction (e.g., amino, carboxy, hydroxy etc.,) may beprotected by methods standard in peptide chemistry prior to reactions ofother groups and subsequently deprotected.

The intermediates of use in the production of the end-products areeither known compounds or can be made by known methods, as described inthe Examples.

The following description of the preparative methods indicates generallythe routes which may be used for the production of the compoundsaccording to the invention.

PROCESS 1, ROUTE A

This process involves reductive amination ##STR14## A keto acid (orderivative) of formula II is condensed with a peptide of formula III.This condensation is conveniently carried out in a suitable solvent(e.g. aqueous tetrahydrofuran, methanol) at a pH between 6 and 7 in thepresence of sodium cyanoborohydride which effects reduction to give thedesired compound of formula I. Alternatively, II and III may be reactedin the solvent medium to form a Schiff's Base as an intermediate andthis may then be reduced catalytically to yield the desired compound offormula I for example by hydrogenation in the presence of Raney Nickelor palladium on charcoal.

As an alternative to Process 1, Route A, the compound of formula II canbe condensed with an amino acid of formula IV below (or protectedderivative thereof) under the same conditions as given in Process 1 toyield an intermediate of formula V. This intermediate is thensubsequently coupled with an amino acid or peptide derivative of theformula A³ to give the compound of formula I. ##STR15## The knownprocesses for peptide bond formation referred to above and also in thefollowing processes encompass reactive group protection during thecoupling reaction, e.g., for amines employing N-t-butyloxycarbonyl orN-benzyloxycarbonyl followed by their removal. Condensing agents aretypically those useful in peptide chemistry such asdicyclohexylcarbodiimide, water soluble carbodiimide [N-ethyl-N¹-(3-dimethylaminopropyl)-carbodiimide], diphenyl phosphoryl azide or Vmay be activated via the intermediary of active esters such as thosederived from 1-hydroxybenzotriazole, 4-nitro phenol, 4-picolyl alcohol.

PROCESS 1 ROUTE B (where R⁴ =H)

In an alternative reductive amination process as shown below, thestarting materials providing the groups A¹ -A² on the one hand and thegroup A³ on the other are reversed. Otherwise the process is the same asProcess 1 Route A. This process is applicable to the production ofcompounds in which R⁴ =H. ##STR16## The amino acid (or derivative) VI iscondensed with the ketone (VII) under the conditions given in Route A.

As an alternative to Process 1, Route B the synthesis can be performedin a step wise manner by condensing VI with the keto acid (orderivative) VIII to yield the intermediate IX. By known processes(summarised above), IX can then be condensed with an amino acid orpeptide derivative A³ to give I. ##STR17##

PROCESS 2 ROUTE A

This process is essentially an alkylation. ##STR18## In this process thepeptide III is alkylated with the appropriate α-haloacid (or derivative)X or α-sulphonyloxy acid in a suitable solvent (e.g., CH₂ Cl₂, CH₃ CN,etc.) in the presence of a base (e.g. triethylamine).

As an alternative to this process, the synthesis can be performed in astepwise fashion firstly to produce an intermediate IX which is thencondensed by standard processes above with a peptide derivative A³ togive the compound of formula I, as described above for the alternativefor Process 1, Route A. ##STR19##

PROCESS 2 ROUTE B

In an alternative alkylation shown below the starting materialsproviding the groups A¹ -A² -- on the one hand and A³ on the other arereversed. Otherwise the method is the same as Process 2, Route A.##STR20## The amino acid (or derivative) VI is alkylated with theα-haloacetyl or α-sulphonyloxyacetyl peptide derivative XI under theconditions described in Route A.

As an alternative to Process 2, Route B the synthesis can be performedin a stepwise fashion by condensing an amino acid (or derivative) VIwith a substituted α-haloacetic acid or α-sulphonyloxy acetic acid (XII)to yield the intermediate IX which by standard processes is condensedwith a peptide derivative A³ to give the compound of formula I ##STR21##

It should be noted that when A¹ and/or A² represent amino acid residues,that these residues may be introduced by standard coupling procedures atany convenient stage of the synthesis.

The starting materials which are required for the above processes areeither known in the literature, or can be made by standard processesfrom known starting materials, or are described in the Examples.

When R¹ in I represents hydroxy, these compounds may be derived fromthose described above (wherein R¹ =alkoxy or aralkoxy) by hydrolysis ina suitable solvent (such as aqueous methanol) containing a base such assodium or lithium hydroxide. Alternatively, when R¹ =aralkoxy (such asPhCH₂ O), this group may be removed by hydrogenolysis.

As mentioned above there are various potentially asymmetric centres inthe amide derivatives of this invention. In particular the carbon atomwhich bears the groups (CH₂)_(n), COR¹ and NH is asymmetric as is thatwhich bears the groups NH, COA³, R² and R³ (when R² and R³ are notsimultaneously hydrogen). The above synthesis can utilize racemates,enantiomers or diastereoisomers or starting materials, the products cantherefore exist in racemic or optically active forms. The inventiontherefore encompasses the racemic form as well as any other opticallyactive forms. As noted above, however, and in contrast to inhibitors ofother zinc metalloproteinases (such as angiotensin converting enzyme),the preferred isomer has R-stereochemistry at the carbon atom bearingthe groups (CH₂)_(n), COR¹ and NH whilst having the stereochemistry ofthe natural amino acids at the other asymmetric centres.

The compounds according to the invention include pharmaceuticallyacceptable salts of the compounds of formula I. Such salts may includeacid addition salts as well as amine salts, etc., and the processesdescribed above for the production of the compounds may include as afinal step the conversion of the compound I into such a salt, or thecompound may be isolated as such salt.

It is understood that the compounds which bind most effectively tocollagenase have R¹ equal to either hydroxy or hydroxyamino. When R¹ isalkoxy or aralkoxy, these compounds function as orally active prodrugsof the parent carboxylic acids; once absorbed these esters are rapidlyhydrolysed by non specific plasma esterases to yield the active species.

In order that the invention may be more fully understood the followingExamples are given by way of illustration and should not limit theinvention in spirit or scope. All temperatures herein are degrees C.

EXAMPLE 1 N-(1-Methoxycarbonylethyl)-L-leucyl-L-valine N-Hexylamide

N-(Tertiarybutyloxycarbonyl)-L-leucyl-L-valine N-Hexylamide (2 g) wastreated with trifluoroacetic acid (20 ml) at room temperature forforty-five minutes. The excess trifluoroacetic acid was removed in vacuoand the residue dissolved in methanol (20 ml). The solution was adjustedto pH7 with triethylamine. Dried 3 A molecular sieve (10 g), sodiumcyanoborohydride (0.75 g) and methyl pyruvate (1.5 g) were added and thereaction mixture stirred at room temperature for 2 days. The reactionmixture was then filtered and the filtrate concentrated in vacuo to agum. The residue was taken up in dichloromethane and the organic phasewashed in turn with saturated sodium hydrogen carbonate solution andthen 1M citric acid solution and dried over sodium sulphate. Thematerial isolated after evaporation of the dichloromethane waschromatographed on silica, developed in a gradient of 20% ethyl acetatein hexane to 60% ethyl acetate in hexane. Elution with 40% ethyl acetatehexane afforded N[1-(S)-methoxycarbonylethyl]-L-leucyl-L-valineN-hexylamide (0.4 g), which crystallised from methanol/water as needlesm.p. 70°-71° C.; [α]_(D) ²⁰ =-31.4° (c=0.2, MeOH); (Found: C,63.0;H,10.2; N,10.5. C₂₁ H₄₁ N₃ O₄ requires C,63.1; H,10.3; N,10.5%); λ_(max)(Nujol): 3400, 1740 and 1610 cm⁻¹ ; (a) δ (CDCl₃) 0.9 (15H, m,2×CH(CH₃)₂ and CH₂ CH₃); 1.3(3H, d, J=6 Hz, CH₃ CH); 1.2-2.4 (12H, m,2×CH(CH₃)₂, CHCH₂ CH and (CH₂)₄); 3.0-3.4 (5H, m, 3×CH and CH₂ NH); 3.7(3H, s; CH₃ --O), 4.3(1H, t, J=8 Hz, NH); 6.94 (1H,m,NH) and 7.85 (1H,d, NH).

Elution with 50% ethyl acetate hexane affordedN[1-(R)-methoxycarbonylethyl]-L-leucyl-L-valine N-hexylamide, (0.5 g)which crystallised from methanol/water as needles m.p. 98°-101° C.;[α]_(D) ²⁰ =-43° (C=0.2, MeOH); (Found: C,62.7; H,10.2; N,10.5. C₂₁ H₄₁N₃ O₄ requires C,63.1; H,10.3; N,10.5%); ν_(max) (Nujol) 3250, 3060,1730 cm⁻¹ ; δ(CDCl₃) 0.9 (15H,m, 2×(CH(CH₃)₂ and CH₂ CH₃); 1.3 (3H,d,J=6Hz, CH₃ CH); 1.2-2.4 (12H,m, 2×CH(CH₃)₂, CHCH₂ CH and (CH₂)₄); 3.0-3.3(4H,m, 2×NHCHCO, CH₂); 3.44(1H,q,J=7 Hz, val α-CH); 3.7(3H,s CH₃ --O);4.28 (1H, q, J=7 Hz,NH); 7.16 (1H,m,NH); and 7.92 (1H,d, J=8 Hz,NH).

The N-(t-butyloxycarbonyl)-L-leucyl-L-valine N-hexylamide used as astarting material was prepared as follows:

N-Tertiarybutyloxycarbonyl-L-valine N-hexylamide (15 g) indichloromethane (30 ml) was treated with trifluoroacetic acid (30 ml) atroom temperature for 45 minutes. The excess trifluoroacetic acid wasremoved in vacuo and the residue redissolved in dichloromethane. Thesolution was adjusted to pH7 with triethylamine,N-tertiarybutyloxycarbonyl-L-leucine (13 g), 1-hydroxybenzotriazole (7g) and DCC (10 g) were added and the reaction mixture stirred at roomtemperature overnight. The reaction mixture was filtered and the organicphase washed with aqueous sodium hydrogen carbonate, 1M citric acid andthen water, dried over sodium sulphate and concentrated in vacuo to agum. The gum was chromatographed on silica developed in a gradient of20% ethyl acetate to 50% ethyl acetate in petrol to affordN-tertiarybutyloxycarbonyl-L-leucyl-L-valine N-hexylamide (19 g) whichcrystallised from ether hexane as needles; m.p. 115°-116° C.; (Found:C,63.2; H,10.3; N,10.1. C₂₂ H₄₃ N₃ O₄.1/4H₂ O requires C,63.2; H,10.5; N10.1%); ν_(max) (nujol) 3300, 3080, 1680, 1630 and 1520 cm⁻¹ ; δ(CDCl₃)0.9 (15H,m, 2×CH(CH₃)₂ and CH₂ CH₃); 1.1-2.3 (12H,m, (CH₂)₄, CH₂CH(CH₃)₂, CHCH(CH₃)₂); 1.45 (9H,s,C(CH₃)₃); 3.25 (2H,m,NHCH₂); 4.12(1H,m, α-CH from leucyl residue); 4.2 (1H,t, J=5 Hz., α-CH from valylresidue); 5.07 (1H,m,NH); 6.55 (1H,m,NH) and 6.80 (1H,d,J=10 Hz, NH).

The N-t-butyloxycarbonyl-L-valine N-hexylamide required as a startingmaterial in the preparation above was synthesised as follows:

Tertiarybutyloxycarbonyl-L-valine (25 g) in dichloromethane (200 ml) wastreated with 1-hydroxybenzotriazole (15.5 g) hexylamine (11.6 g) and DCC(26 g) at room temperature for 2 days. The solution was filtered and theorganic phase washed with aqueous sodium hydrogen carbonate, aqueouscitric acid (1M) and water, dried over sodium sulphate and concentratedin vacuo to afford tertiary butyloxycarbonyl-L-valine N-hexylamide (28g) which crystallised from methanol-water as needles; m.p. 74°-76° C.;(Found: C,63.8; H,10.6; N,9.4. C₁₆ H₃₂ N₂ O₃ requires C,64.0; H,10.7;N,9.32%); ν_(max) (Nujol): 3280 and 1630 cm⁻¹ ; δ(CDCl₃) 0.8-1 (9H,m,3×CH₃); 1.3 (8H,m,(CH₂)₄); 1.45 (9H,s, (CH₃)₃ C); 2.1 (1H,m,CH(CH₃)₂);3.3 (2H,m, NHCH₂ ); 3.9 (1H,dd, J=8 Hz and 5 Hz., α-CH); 5.2 (1H,d,J=8Hz, CONH) and 6.26 (1H,m,NH).

EXAMPLE 2 N-[1-(R)-Methoxycarbonylethyl]-L-leucyl-O-benzyl-L-tyrosineN-methylamide

N-Boc-O-benzyl-L-tyrosine methylamide (3 g, 7.7 mM) was dissolved in 1:1TFA/CH₂ Cl₂ (100 ml). After 15 min. the solvent was removed in vacuo andthe residue taken up in H₂ O (100 ml), neutralised with NaHCO₃ andextracted into CH₂ Cl₂ (3×100 ml). The organic extract was dried andevaporated in vacuo to yield a white solid (2.2 g). This material in CH₂Cl₂ (50 ml) and DMF (5 ml) was treated at 0° withN-[1-(R)-methoxycarbonylethyl]-L-leucine (1.3 g, 6 mM),1-hydroxybenzotriazole (960 mg, 6.4 mM) and dicyclohexylcarbodiimide(1.3 g, 6.5 mM) and the mixture allowed to warm to room temperature over2 h. After a further 12 h the reaction mixture was filtered, washed withsat. NaHCO₃ and then brine, dried and then evaporated in vacuo to yielda solid, 2.5 g (68%). Recrystallisation from CH₂ Cl₂ /hexane gave thetitle compound; m.p. 65°-68°; [α]_(D) ²⁰ =-3.3° (C=0.2, MeOH); (Found:C, 66.72; H, 7.61; N, 8.72. C₂₇ H₃₇ N₃ O₅ requires C, 67.02; H, 7.71; N,8.69%); ν_(max) (nujol) 3280 br, 1735, 1635 and 1510 cm⁻¹ ; δ(CDCl₃)0.87 and 0.9 (each 3H, each d, J=4 Hz. and 2.5 Hz, (CH₃)₂ CH); 1.1(1H,m,(CH₃)₂ CH₂ CH); 1.3 (3H,d,J=8.5 Hz., CH₃ CH); 1.45 (2H,m, (CH₃)₂ CH₂CH); 1.58 br (1H,s, CHNHCH, exch); 2.77 (3H,d,J=6 Hz, NHCH₃); 3.0(1H,dd,J=12 and 8 Hz, CH₂ C₆ H₄); 3.07 (1H,m, (CH₃)₂ CH₂ CH); 3.18(1H,dd,J=12 and 6 Hz, CHCH₂ C₆ H₄); 3.38 (1H,q,J=8.5 Hz, CH₃ CH); 3.68(3H,s,OCH₃), 4.62 (1 H,q,J=7 Hz, NHCH(CH₂ C₆ H₄)CO); 5.02 (2H,s, OCH₂ C₆H₅); 6.71 br (1H,q,J ca 5 Hz, exch. NHCH₃); 6.89 and 7.12 (each 2H, eachd, each J=8 Hz, C₆ H₄); 7.4 (5H,m,C₆ H₅) and 7.75 (1H,d, J=9 Hz, exch,CONHCHCO); m/e 484 (100%, [M+1]⁺), 381 (27) and 172 (28).

The syntheses for the two starting materials required in the preparationabove are described in the following paragraphs.

(a) N-t-Butyloxycarbonyl-O-benzyl-L-tyrosine N-Methylamide

N-t-Butyloxycarbonyl-O-benzyl-L-tyrosine (7.4 g, 20 mM),1-hydroxybenzotriazole (3 g, 20 mM), methylamine hydrochloride (1.3 g,20 mM) and N-methyl morpholine were dissolved in CH₂ Cl₂ (200 ml) andcooled to 0° C. Dicyclohexylcarbodiimide (4.2 g, 20 mM) was added andthe reaction allowed to warm to room temperature over 4 h. After afurther 12 h the reaction mixture was filtered; the filtrate was washedwith sat NaHCO₃, 3N citric acid and brine, dried and evaporated in vacuoto give the required N-methylamide which was recrystallised from CH₂ Cl₂and hexane (4.5 g, 58%); m.p. 165°-172°; [α]_(D) ²⁰ =+15.2° (C=0.2,MeOH); (Found: C, 68.85; H, 7.43; N, 7.39. C₂₂ H₂₈ N₂ O₄ requires C,68.73; H, 7.34; N, 7.29%); ν_(max) (nujol) 3330, 1685, 1672, 1655 and1520 cm⁻¹ ; δ(CDCl₃) 1.4 (9H,s, (CH₃)₃ C); 2.91 (3H,d, J=5 Hz, NHCH₃);3.0 (2H,m,CH₂ C₆ H₄); 4.26 (1H,q, J=7.5 Hz, NHCH(CH₂)CO); 5.04 (2H,s,OCH₂ C₆ H₅); 5.08 br (1H,s, exch, NH); 5.84 br (1H,s,exch); 6.91 and7.09 (each 2H, each d, each J=8 Hz., C₆ H₄) and 7.4 (5H,m,C₆ H₅); m/e385 (68%, [M+1]⁺), 329 (100), 285 (66) and 267 (58).

(b) N-[1-(R)-Methoxycarbonylethyl]-L-luecine

This was prepared in two steps from L-leucine benzyl ester asillustrated below:

L-Leucine benzyl ester, para-toluene sulphonic acid salt (120 g, 0.3M)was dissolved in dry methanol (300 ml) and the pH (moist pH paper)adjusted to 6 using Et₃ N and acetic acid. Methyl pyruvate (62.4 g,0.6M) in dry methanol (10.0 ml) and 3 A molecular sieves were added; themixture was cooled to 5° and then NaBH₃ CN (100 g, 1.58M) in methanol(600 ml) added. After stirring for 3 days the reaction mixture wasfiltered and evaporated in vacuo. The residual white solid waspartitioned between H₂ O (500 ml) and CH₂ Cl₂ (4×200 ml); the organicphase was evaporated to a yellow oil and then partitioned between hexane(250 ml) and 1M oxalic acid (4×250 ml). The aqueous phase wasneutralised with NaHCO₃ and extracted into CH₂ Cl₂ (4×250 ml). Theorganic phase was dried and evaporated in vacuo to yield a yellow oil(90 g), which was chromatographed on SiO₂ using a gradient of EtOAc inhexane as eluant. The faster running diastereoisomer,N-[1-(R)-methoxycarbonylethyl]-L-leucine benzyl ester, was isolated asan oil (22 g, 20%); [α]_(D) ²⁰ =-49.5° (C=0.2, MeOH); (Found: C, 66.06;H, 8.19; N, 4.75. C₁₇ H₂₆ NO₄ requires C, 66.42; H, 8.19; N, 4.54);ν_(max) (nujol) 1735 cm⁻¹ ; δ(CDCl₃) 0.89 and 0.92 (each 3H, each d,each J=3.5 Hz., (CH)₃)₂); 1.29 (3H,d,J=7 Hz; CH₃); 1.5 (2H,m,CH₂ CH):1.74 (2H,m,NH and CH₂ CH(CH₃)₂); 3.34 (1H,q, J=7 Hz, CHCH₃), 3.39(1H,t,J=7 Hz, CH₂ CH(NH)CO); 3.69 (3H, s, OCH₃); 5.15 (2H,m,OCH₂ C₆ H₅)and 7.35 (5H,s,C₆ H₅); m/e 308 (100%; [M+1]⁺); 232 (53) and 172 (44).

The slow running diastereoisomer,N-[1-(S)-methoxycarbonylethyl)-L-leucine benzyl ester, was isolated asan oil (11.3 g, 10%); [α]_(D) ²⁰ =1.73° (C=0.2, MeOH); (Found: C, 66.42;H, 8.30; N, 4.54. C₁₇ H₂₆ NO₄ requires C, 66.42; H, 8.19; N, 4.55%)Γ_(max) (film) 1730 cm⁻¹ ; δ(CDCl₃) 0.87 and 0.9 (each 3H, each d, eachJ=5.5 Hz, (CH₃)₂ CH); 1.27 (3H,d,J=7 Hz, CH₃ CH); 1.49 (2H,m, (CH₃)₂--CHCH₂); 1.74 (1H, heptet, J=7 Hz, (CH₃)₂ CH); 2.2 br (1H,s,NH), 3.3(2H,m,CHNHCH), 3.65 (3H,s,OCH₃); 5.13 (2H,s,OCH₂ C₆ H₅) and 7.35(5H,s,C₆ H₅); m/e 308 (100%, [M+1]⁺) and 172 (100).

The R-benzyl ester (13 g, 42 mM) was dissolved in methanol (300 ml) andhydrogenated over 10% palladium on charcoal at atmospheric pressure. Thecatalyst was removed by filtration through celite and the filtrateevaporated in vacuo to yield a white gum, which was crystallised fromMeOH/Et₂ O to give the required leucine derivative as a whitecrystalline solid (7.5 g, 82%); mp 150°-151°; (Found: C, 55.27; H, 8.72;N, 6.43. C₁₀ H₁₉ NO₄ requires C, 55.3; H, 8.81; N, 6.45%); [α]_(D) ²⁰8.4 (C=0.2, MeOH); ν_(max) (nujol) 3400 br, 2500 br and 1755 cm⁻¹ ; δ(d⁶DMSO) 0.85 (6H, m, (CH₃)₂ CH₂); 1.17 (3H,d,J=7 Hz, CH₃ CH); 1.38 (2H,m,(CH₃)₂ CHCH₂); 1.74 (1H, heptet, J=6 Hz, (CH₃)₂ CH); 3.14 (1H, t, J=7Hz, NHCH(CH₂)CO₂ H); 3.29 (1H,q,J=7 Hz, CH₃ CH) and 3.6 (3H,s,OCH₃); m/e218 (100%, [M+1]⁺), 172 (27) and 158 (17).

EXAMPLE 3N-[2-N-[N-(2,4-Dinitrophenyl)-L-prolyl-L-leucyl]amino-1-(R)-methoxycarbonylethyl]-L-leucyl-O-benzyl-L-threonineN-Methylamide

This was prepared starting from methylN-t-butyloxycarbonyl-N-benzyloxycarbonyl(R)-2,3-diaminopropionate andbenzyl 4-methyl-2-oxo-pentanoate in the steps described in the followingparagraphs.

(a)N-[2-N-(t-Butyloxycarbonyl)amino-1-(R)-methoxycarbonylethyl]-L-leucineBenzyl ester

To a stirred solution of methyl N-t-butyloxycarbonyl-N-benzyloxycarbonyl-(R)-2,3-diaminopropionate (25 g) in THF (150 ml) and aceticacid (8 ml) was added palladised charcoal (10%, 2 g) and the mixturehydrogenated at 25° and 760 mmHg for 2 h. The catalyst was removed byfiltration and to the filtrate was added THF (50 ml), benzyl4-methyl-2-oxopentanoate (50 g, from the corresponding acid by treatmentat reflux with benzyl alcohol in the presence of para-toluene sulphonicacid and azeotropic removal of water) in THF (50 ml) and finally water(70 ml). The pH of the rapidly stirred solution was adjusted to 6.5 withtriethylamine and sodium cyanoborohydride (4.5 g) was added portionwiseover 0.5 h. The pH was maintained at 6.5 by periodic addition of aceticacid. After 16 h at 20°, a further portion of sodium cyanoborohydride (2g) was added and stirring continued for 24 h. The reaction mixture wasconcentrated in vacuo and the residue was partitioned between CH₂ Cl₂(200 ml) and water (100 ml). The aqueous layer was washed with fresh CH₂Cl₂ (2×100 ml) and the combined organic extracts washed successivelywith 3N-citric acid solution water and finally saturated aqueous sodiumhydrogen carbonate solution and then dried over MgSO₄. The oil isolatedfrom the CH₂ Cl₂ was purified by chromatography on silica eluting withCH₂ Cl₂ in an increasing ethyl acetate gradient to give the requiredbenzyl ester (9.6 g) as an oil which slowly crystallised, m.p. 59.5°-61°(from etherhexane); [α]_(D) ²⁵ =22.1° (C=1.1, MeOH); (Found: C, 62.40;H, 8.08; N, 6.57. C₂₂ H₃₄ N₂ O₆ requires C, 62.54; H, 8.11; N, 6.36%);ν_(max) (CHCl₃) 1730 and 1705 cm⁻¹ ;δ (CDCl₃) 0.89 (6H, t, J=6.3 Hz,CH(CH₃)₂); 1.43 (9H,s, C(CH₃)₃); 1.50 (2H, m, CH₂ CH); 1.76 (2H,m,CH₂CH(CH₃)₂ and NH); 3.35 (4H,m,CH₂ N and 2×α-CH); 3.67 (3H,s,OCH₃); 4.98br (1H,s,NHCOO), 5.12 (2H,d, J=11.5 Hz, CH₂ Ph) and 7.36 (5H, m,C₆ H₅);m/e 423 ([M-1]⁺).

(b)N-[2-N-(t-Butyloxycarbonyl)amino-1-(R)-methoxycarbonylethyl]-L-leucyl-O-benzyl-L-threonineN-Methylamide

The foregoing benzyl ester (6 g) in methanol (50 ml) was hydrogenated atS.T.P. over 10% palladised charcoal (100 mg) for 0.5 h. The catalyst wasremoved by filtration and the material recovered from the methanol wasrecrystallised from methanol-ether to give the intermediate carboxylicacid (4.5 g),

m.p. 147°-148°. A portion of this material (2.8 g) in CH₂ Cl₂ (100 ml)and DMF (20 ml) was treated at 0° with 1-hydroxybenzotriazole (1.3 g),and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.61g). After 0.5 h at 0°, L-O-benzyl-threonine N-methylamide (1.86 g) inCH₂ Cl₂ (10 ml) was added and the mixture allowed to warm to 20° over 1h. After 36 h at 20°, the reaction mixture was washed in turn withsaturated sodium hydrogen carbonate solution, 3N-citric acid solutionand finally brine and then dried and evaporated in vacuo.Crystallisation of the resulting oil from ether-pentane gaveN-[2-N-(t-butyloxycarbonyl)amino-1-(R)-methoxycarbonylethyl]-L-leucyl-O-benzyl-L-theronineN-methylamide (3 g), m.p. 95°-97°; (Found; C, 60.42; H, 8.20; N, 10.44.C₂₇ H₄₄ N₄ O₇ requires C, 60.43; H, 8.26; N, 10.44%); δ (CDCl₃) 0.94(6H,d, J=6.2 Hz., CH(CH₃ )₂); 1.13 (3H,d,J=6.4 Hz, CHCH₃); 1.43(9H,s,C(CH₃)₃); 1.54-1.85 (3H,m,CH₂ CH); 1.95 broad (1H,s, NH); 2.82(3H,d, J=4.8 Hz, NHCH₃); 3.1-3.62 (4H,m,NCH₂, OCH and αCH), 3.65(3H,s,OCH₃), 4.3 (1H,m,α-CH), 4.45 (1H,dd, J=6.3 and 2.3 Hz, α-CH); 4.54and 4.62 (each 1H, each d, each J=11.6 Hz, CH₂ Ph); 5.05 broad (1H,s,NHCOO), 7.05 (1H,m,NHCH₃), 7.32 (5H,m,C₆ H₅) and 7.88 (1H,d, J=8.4 Hz,NH).

(c)N-[2-N-[N-(2,4-Dinitrophenyl)-L-prolyl-L-leucyl]amino-1-(R)-methoxycarbonylethyl]-L-leucyl-O-benzyl-L-threonineN-Methylamide

To a stirred solution of the foregoing t-butyloxycarbonyl protectedpeptide (536 mg) in CH₂ Cl₂ (3 ml) was added trifluoroacetic acid (3 ml)at 0°. The solution was allowed to warm to 20° and then stirred at thistemperature for 2 h. The residue after evaporation of the organicsolvents was taken into CH₂ Cl₂ and the solution washed with saturatedsodium hydrogen carbonate solution, dried (Na₂ SO₄) and evaporated invacuo to yieldN-[2-amino-1-(R)-methoxycarbonylethyl]-L-leucyl-O-benzyl-L-threonineN-methylamide (330 mg). This material in CH₂ Cl₂ (10 ml) was added to asolution of N-[N-(2,4-dinitrophenyl)-L-prolyl]-L-leucine (330 mg) in CH₂Cl₂ (10 ml) containing 1-hydroxybenzotriazole (132 mg) andN-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (191 mg)stirred at 5°. After 16 h at 4° the solvent was removed in vacuo and theresidue in ethyl acetate washed in turn with water, saturated sodiumhydrogen carbonate solution and finally 3N-citric acid solution. Thematerial isolated from the ethyl acetate was recrystallised from CH₂ Cl₂-- ether to give the required peptide (440 mg), m.p. 138°-142°; (Found:C, 57.34; H, 6.92; N, 13.61. C₃₉ H₅₆ N₈ O₁₁ requires C, 57.62; H, 6.94;N, 13.78%); ν_(max) (CHCl₃) 3295, 1730 and 1635 cms⁻¹ ; δ(CDCl₃) 0.95(12H,m, 2×CH(CH₃)₂); 1.14 (3H,d,J=6.3 Hz, CH₃ CH); 1.3-2.15 and 2.45(10H,m,CH₂ CH₂ and 2×CH₂ CH); 2.74 (3H,s,NHCH₃); 3.3 (3H,m,CH₂ N andCHO); 3.56 (3H,m,CH₂ N and α-CH); 3.63 (3H,s,OCH₃); 4.06, 4.25 and 4.56(1H,2H and 1H respectively, each m, 4×αCH); 4.43 and 4.55 (each 1H, eachd, J=11.7 Hz, CH₂ Ph); 7.00 (1H,d, J=9.5 Hz, 6-H in C₆ H₃); 7.28(5H,s,C₆ H₅); 8.16 (1H,dd, J=9.5 and 2.8 Hz, 5-H in C₆ H₃) and 8.54(1H,d, J=2.8 Hz, 3-H in C₆ H₃); m/e 813 ([M+1]⁺).

O-Benzyl-1-threonine N-methylamide used in step (b) above was preparedfrom N-t-butyloxycarbonyl-O-benzyl-L-threonine N-methylamide by exposureto trifluoroacetic acid in CH₂ Cl₂. This in turn was prepared fromN-t-butyloxycarbonyl-O-benzyl-L-threonine and methylamine using theprocedure described in Example 2 for the tyrosine analogue.

N-[N-(2,4-Dinitrophenyl)-L-prolyl]-L-leucine used as starting materialin stage (c) was prepared from N-(2,4-dinitrophenyl)-L-proline andleucine methyl ester using the coupling procedure involvingN-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride as thecondensing agent in the presence of 1-hydroxybenzotriazole (asillustrated in Example 3) followed by hydrolysis of the methyl esterwith 2N-sodium hydroxide solution (see Example 5).

MethylNβ-t-butyloxycarbonyl-Nα-benzyloxycarbonyl-(R)-2,3-diaminopropionate,used as the starting material in stage (a), was prepared as follows:

To a stirred suspension of N-benzyloxycarbonyl-(R)-2,3-diaminopropionicacid [19.5 g; from Nα-benzyloxycarbonyl-D-asparagine exactly asdescribed for the L-isomer in Synthesis, 266, (1981)] in dry methanol(60 ml) at -20° was added thionyl chloride (30 g) dropwise over 40 min.The reaction mixture was allowed to warm to 20° over 1 h and then heatedat 50° for 1 h. The residue after removal of the solvent wasrecrystallised from methanol-ether to give methylNα-benzyloxycarbonyl-R-2,3-diaminopropionate hydrochloride (22.5 g);m.p. 170°-172°; (Found: C, 49.86; H, 5.89; N, 9.53. C₁₂ H₁₇ N₂ O₄ Clrequires C, 49.91; H, 5.93; N, 9.70%); ν_(max) (Nujol) 3305, 1735 and1688 cm⁻¹ ; δ (d⁶ DMSO) 3.00-3.32 (2H,m,CH₂ NH₂); 3.7 (3H,s,OCH₃); 4.45(1H,m,α-CH); 5.09 (2H,s,CH₂ Ph); 7.36 (5H,s,C₆ H₅); 7.95 (1H,d,J=7.5 Hz,NHCOO) and 8.28 broad (3H,s, NH₃); m/e 253 ([M+1]⁺). A portion of thismaterial (22.5 g) in DMF (150 ml) was treated with Et₃ N until the pHwas 10. Di-t-butyl dicarbonate (16.8 g) was added to the solutionstirred at 5°. After a further 2 h at 20°, the reaction mixture wasfiltered and evaporated in vacuo and the residue partitioned betweenether and water. The aqueous layer was extracted twice more with freshether and the combined organic extracts washed in turn with ice cold1N-hydrochloric acid, saturated sodium hydrogen carbonate solution andfinally water. The oil isolated from the ether was crystallised fromethyl acetate-hexane to give methylNβ-t-butyloxycarbonyl-Nα-benzyloxycarbonyl-(R)-2,3-diaminopropionate(22.5 g); m.p. 89°-91°; (Found: C, 57.86; H, 6.95; N, 7.93. C₁₇ H₂₄ N₂O₆ requires C, 57.94; H, 6.86; N, 7.95%); ν_(max) (CHCl₃) 3600 and 1700cm⁻¹ ; δ 1.4 (9H,s, C(CH₃)₃); 3.5 broad (2H, s, CH₂ N); 3.72 (3H, s,OCH₃); 4.4 (1H, α-CH); 5.09 (2H, s, CH₂ Ph); 5.2 broad (1H,s, NHCOO);6.06 (1H, d, J=7.3 Hz, NHCOO) and 7.32 (5H, s, C₆ H₅).

EXAMPLE 4 N-[1-(R)-Methoxycarbonylethyl]-L-leucyl-O-benzyl-L-tyrosineN-Methylamide

Leucine benzyl ester para-toluene sulphonic acid salt (113 g) in dryacetonitrile (800 ml) was treated with methyl 2-bromopropionate (62.7ml) and N-methyl morpholine (100 ml) under reflux for 16 h. The reactionmixture was concentrated in vacuo and the residue in ethyl acetatewashed with brine, dried (Na₂ SO₄) and evaporated. Chromatography of theresulting oil on silica in 1:4 ethyl acetate-hexane gaveN-[1-(R)-methoxycarbonylethyl]-L-leucine benzyl ester (45 g) as thefaster running fraction. This material was treated exactly as describedabove in Example 2 to give the title compound.

EXAMPLE 5 N[1-(R)-Carboxyethyl]-L-leucyl-O-benzyl-L-tyrosineN-Methylamide

The methyl ester (1.5 g, 3.1 mM) from Example 2 was dissolved inmethanol (20 ml) and treated with 1N sodium hydroxide (3.5 ml, 3.5 mM).After 18 h, the pH was adjusted to 5 with acetic acid and the solventremoved in vacuo to yield a white solid. Recrystallisation first fromwater and then from methanol-ether yielded theN-[1-(R)-carboxyethyl]-L-leucyl-O-benzyl-L-tyrosine N-methylamide as awhite powder (1.02 g), m.p. 195°; [α]_(D) ²⁰ =+7.2° (C=0.2, MeOH);(Found: C, 63.76; H, 7.64; N, 8.57. C₂₆ H₃₅ N₃ O₅.H₂ O requires C,64.05; H, 7.65; N, 8.62%); ν_(max) (Nujol) 3540 (br), 3330 and 1680 cm⁻¹; δ (d⁶ DMSO) 0.82 (3H,d, J=6 Hz, (CH₃)₂ CH); 0.87 (3H,d, J=6 Hz, (CH₃)₂CH); 1.13 (3H,d, J=7 Hz., CH₃ CH); 1.24 (2H,t, J=6 Hz., CHCH₂ CH: 1.59(1H,m, (CH₃)₂ CHCH₂); 2.63 (3H,d, J=5 Hz, NHCH₃); 2.72 (1H,dd, J=11 and12 Hz, CHCH₂ C₆ H₄); 2.8 (1H,q, J=7 Hz, CH₃ CH(NH)CO₂ H); 2.95 (1H,dd,J=12 and 5 Hz, CHCH₂ C₆ H₄); 3.21 (1H,t, J=7.5 Hz, NHCH(CH₂ CH(CH₃)₂CO); 4.53 br (1H,m, NHCH(CH₂ C₆ H₄)CO); 5.08 (2H,s,C₆ H₄ OCH₂ C₆ H₅);6.93 and 7.17 (each 2H, each d, each J=7.5 Hz, C₆ H₄ O); 7.48 (5H,m,C₆H₅); 7.98 br (1H,q, J=5 Hz, NHCH₃, exch) and 8.1 (1H,d, J=9 Hz.,CHCONHCH, exch); m/e 470 (88% [M+1]⁺ ), 452 (51), 424 (29), 285 (100)and 158 (49).

EXAMPLE 6 N[1-(R)-Carboxyethyl]-L-Leucine N-Phenethylamide

N[1-(R)-Ethoxycarbonylethyl]-L-Leucine N-phenethylamide (710 mg, 2.1 mM)was dissolved in MeOH (50 ml) and treated with IN NaOH (3 ml, 3 mM) atroom temperature. After 12 h, the reaction mixture was acidified withAcOH and evaporated in vacuo to a solid which was washed with H₂ O anddried to yield the title compound (400 mg); m.p. 201°-205°; (Found: C,66.44; H, 8.55; N,9.11; C₁₇ H₂₆ N₂ O₃ requires C,66.64; H,8.55;N,9.14%); ν_(max) (Nujol) 3330, 1660 and 1530 cm⁻¹ ; δ (d⁶ DMSO) 0.825(6H,t,J=6.2 Hz, (CH₃)₂ CH), 1.15 (3H,d, J=6.8 Hz, CH₃ CH), 1.29(2H,m,CH₂ CH), 1.55 (1H, heptet,J=7 Hz, CH(CH₃)₂), 2.71 (2H,t,J= 7 Hz,CH₂ C₆ H₅), 3.0 (1H,q,J=7 Hz, CHCH₃), 3.14 (1H,t,J=7 Hz, α-CH), 3.32(2H,q,J=6 Hz, NHCH₂ CH₂), 7.12 (5H,m,C₆ H₅), 7.5 (2H,br S,OH and CHNHCH)and 8.15 (2H,t,J=5 Hz, NHCH₂).

The N[1-(R)-ethoxycarbonylethyl]-L-leucine N-phenethylamide required inthe preparation above was synthesised as follows:

N[1-(R)-Ethoxycarbonylethyl]-L-leucine (1.39 g, 6 mM),N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (1.16 g, 6mM), 1-hydroxybenzotriazole (0.93 g, 6 mM) and phenethylamine (0.7 g, 6mM) were dissolved in DMF (50 ml) at -6°. N-Methyl-morpholine (0.62 g,6.2 mM) was added and the reaction mixture allowed to warm to roomtemperature. After 12 h the solvent was removed in vacuo. The residue inEtOAc (150 ml) was washed with H₂ O (2×100 ml), dried and evaporated invacuo to yield an oil which was purified by chromatography on SiO₂ inEtOAc to give N[1-(R)-ethoxycarbonylethyl]-L-leucine N-phenethylamide asan oil (1.84 g). For analysis, a portion of this material was dissolvedin MeOH, treated with anhydrous HCl in Et₂ O and evaporated in vacuo toyield the corresponding hydrochloride as a foam; (Found: C,60.28;H,8.54; N,7.35; C₁₉ H₃₀ N₂ O₃.HCl.O.4H₂ O requires C,60.35; H,8.48;N,7.41%); [α]_(D) ²⁰ =+9.4° (c=0.2, MeOH); ν_(max) (Nujol) 3400 (br),3100 (br), 2510 (br), 2400 (br), 1740, 1670 and 1550 cm⁻¹ ; δ CDCl₃)0.92 and 0.95 (6H, each d, each J=7 Hz, (CH₃ )₂ CH), 1.27 (3H, t, J=6.5Hz, CH₃ CH₂), 1.28 (3H,d,J=7 Hz, CH₃ CH), 1.3-1.7 (3H,m,CH₂ CH), 2.85(2H,t,J=6 Hz,CH₂ CH₂ C₆ H₅), 3.21 (1H,dd,J=10 Hz and 4 Hz, α-CH), 3.27(3H,q,J=6.5 Hz, CH₃ CH), 3.54 (2H,q,J=7 Hz, NHCH₂ CH₂), 4.16 and 4.18(2H, each q, each J=6.5 Hz, OCH₂ CH₃) and 7.25 (6H,m,C₆ H₅ and NHCO).

The starting material required in the preparation given above wassynthesised in two steps from leucine benzyl ester as follows:

(a) N[1-(R)-Ethoxycarbonylethyl]-L-Leucine Benzylester

L-Leucine benzyl ester (186.65 g, 0.843M), ethyl 2-bromopropionate(153.1 g, 0.846M) and N-methylmorpholine (165 ml, 1.5M) were dissolvedin dry CH₃ CN (800 ml) and refluxed for 12 h. The solvent was removed invacuo and the residue partitioned between H₂ O (21) and EtOAc (3×11).The organic phase was washed with brine, dried and evaporated in vacuo.The resulting oil was chromatographed on SiO₂ in 7.5% EtOAc in hexane togive the title compound (70 g) as the faster running fraction; (Found:C,67.02; H,8.42; N,4.25; C₁₈ H₂₇ NO₄ requires C,67.26; H,8.47; N,4.36);ν_(max) film 1730 cm⁻¹ ; δ (CDCl₃) 0.88 and 0.9 (each 3H, each d, eachJ=6.5 Hz, CH(CH₃)₂) 1.23 (3H,t,J=7 Hz,CH₃ CH₂ O), 1.27 (3H,d,J=7 Hz,CH₃CH), 1.5 (2H,m,CHCH₂), 1.7 (1H, heptet, J=7 Hz, CH(CH₃)₂), 2.2 (1H, brs,NH), 3.32 (2H,m, CHNHCH), 4.10 and 4.12 (each 1H, each q, each J=7 Hz,OCH₂ CH₃), 5.12 (2H,s, OCH₂ C₆ H₅) and 7.34 (5H,s, C₆ H₅); m/e 322(100%; [m+1])⁺, 260 (15), 186 (26) and 112 (28).

(b) N[1-(R)-Ethoxycarbonylethyl]-L-leucine

N[1-(R)-Ethoxycarbonylethyl]-L-leucine benzyl ester (69.09 g, 0.215M)was dissolved in MeOH (300 ml) and hydrogenated at 1 atmosphere over 5%palladium on charcoal (5 g). After 1.5 h, the catalyst was removed byfiltration and the filtrate evaporated in vacuo to yield a solid (46.8g) which was recrystallised from MeOH/Et₂ O to yield the title compound(24 g); m.p. 149°-150°; [α]_(D) ²⁰ =8.8° (C=1.4, MeOH); (Found: C,57.14;H,9.06; N,6.02; C₁₁ H₂₁ NO₄ requires C,57.12; H,9.15; N,6.06%); ν_(max)(Nujol) 3090 (br), 2300 (br), 1755 and 1560 cm⁻¹ ; δ (d⁶ DMSO) 0.86 and0.87 (6H, each d, each J=6.5 Hz, (CH₃)₂ CH), 1.19 (6H,m,OCH₂ CH₃ andCHCH₃), 1.36 (2H,m, CHCH₂ CH), 1.74 (1H, heptet, J=6.5 Hz, CH₂CH(CH₃)₂), 3.14 (1H,t,J=6.2 Hz, δ-CH), 3.27 (1H,q,J=6.8 Hz,NHCHCH₃) and4.07 (2H,q,J=7 Hz,OCH₂ CH₃); m/e 232 (100%, [m+1]⁺), 186 (3) and 158(7).

EXAMPLE 7N[1-(R)-Carboxy-3-methylthiopropyl]-L-leucyl-O-methyl-L-tyrosineN-methylamide

This was prepared from D-methionine methyl ester hydrochloride,2-oxo-4-methylpentanoic acid and O-methyl-2-tyrosine in the stepsdescribed in the following paragraphs.

(a)N[1-(R)-Carbomethoxy-3-methylthiopropyl]-L-leucyl-O-methyl-L-tyrosineN-methylamide

D-Methionine methyl ester hydrochloride (10 g, 50 mM) and2-oxo-4-methylpentanoic acid t-butyl ester (9.3 g, 50 mM) were dissolvedin THF (75 ml) and H₂ O (25 ml). The pH was adjusted to 6.5 withN-methylmorpholine, NaCNBH₃ (630 mg, 10 mM) was added, followed after 2h by a further portion (400 mg). After 18 h the reaction mixture wasevaporated in vacuo and then partitioned between EtOAc (100 ml) andsat.NaHCO₃ solution (2×100 ml). The oil isolated from the organic layerwas chromatographed on SiO₂ using a gradient of 5-10% EtOAc in hexane.The faster running fraction afforded the required isomer as an oil (1.4g). δ (CDCl₃) 0.96 (6H,m, (CH₃)₂ CH), 1.5 (9H,m, (CH₃)₃ C), 1.4-2.0(5H,m, CH₂ CH and SCH₂ CHCH), 2.1 (3H,S,CH₃ S), 2.62 (2H,m, SCH₂ CH₂),3.17 and 3.38 (each 1H, t, each J=7 Hz, CHNHCHO and 3.7 (3H,s,OCH₃)].The slower running isomer was also obtained as an oil (1.2 g). δ (CDCl₃)0.95 (6H,m, (CH₃)₂ CH), 1.5 (9H,s, (CH₃)₃ C), 1.5-2.1 (5H,m, SCH₂ CH₂ CHand CH₂ CH), 2.09 (3H,s,CH₃ S), 2.6 (2H,m,SCH₂ CH₂), 3.08 and 3.22 (each1H, each dd, each J=7 Hz, CHNHCH) and 3.7 (3H,s,OCH₃)].

The faster runnng t-butyl ester (2.9 g, 9 mM) from above was dissolvedin TFA (50 ml) and H₂ O (0.5 ml). After 3 h the mixture was evaporatedin vacuo, toluene (50 ml) was added and the solution was reevaporated invacuo. The resulting oil was dissolved in CH₂ Cl₂ (100 ml) and the pHadjusted to 7 (moist pH paper). O-Methyl-L-tyrosine N-methylamide (2.0g, 10 mM) and 1-hydroxybenzotriazole (1.5 g, 10 mM) were added. Thereaction mixture was cooled to 0°, treated with dicyclohexylcarbodiimide(2.1 g, 10 mM) and then allowed to warm slowly to room temperature.After 18 h, the mixture was filtered and the filtrate washed with H₂ Oand sat. NaHCO₃ solution. After drying, the solvent was removed in vacuoto yield an oil which was chromatographed on SiO₂ in 1:1 EtOAc/hexane.The relevant fractions yielded, after recrystallisation from Et₂O/hexane, the title compound (1.4 g); m.p. 108°-111°; (Found: C,58.62;H,7.91; N,8.85; C₂₃ H₃₅ N₃ O₅ S requires C,59.07; H,7.97; N,8.96%);ν_(max) (Nujol) 3380 (br), 1740, 1610 and 1560 cm⁻¹ ; δ (CDCl₃) 0.86 and0.87 (each 3H, each d, each J=6 Hz, (CH₃)₂ CH), 1.15 and 1.4 (each 1H,each m, CH₂ CH(CH₃)₂), 1.6 (1H,m,CH), 1.90 (2H,m,SCH₂ CH₂), 2.08(3H,s,CH₃ S), 2.5 (2H,m, SCH₂), 2.77 (3H,d,J=6 Hz, NHCH₃), 3.05(3H,m,CH₂ C₆ H₅ and α-CH) 3.47 (1H,t,J=5 Hz, α-CH), 3.7 (3H,s,OCH₃), 3.8(3H,s,CO₂ CH₃), 4.63 (1H,q,J=7 Hz, α-CH), 6.69 (1H,brq,J=6 Hz, NHCH₃),6.82 and 7.13 (each 2H, each d, J=9 Hz, C₆ H₄) and 7.53 (1H,d,J=9 Hz,CONHCH); m/e 684 (100%, [m+1]⁺) and 232 (27).

(b) N[1-(R)-Carboxy-3-methylthiopropyl]-L-leucyl-O-methyl-L-tyrosineN-Methylamide

N[1-(R)-Carbomethoxy-3-methylthiopropyl]-L-leucyl-O-methyl-L-tyrosineN-methylamide (100 mg, 0.2 mM) was dissolved in MeOH (10 ml) and treatedwith IN NaOH (0.25 ml, 0.25 mM). After 18 h another portion of IN NaOH(0.5 ml, 0.5 mM) and H₂ O (2 ml) were added. After a further 18 h thereaction mixture was acidified with AcOH and evaporated in vacuo. Theresulting white solid was chromatographed on C₁₈ -Silica eluting with agradient of 10% to 40% MeOH in H₂ O. The relevant fractions were pooledand evaporated in vacuo; the residue was recrystallised from hot H₂ O toyield the title compound (20 mg); m.p. 170-180; (Found: C,56.88; H,7.49;N,9.05; calculated for C₂₂ H₃₅ N₃ O₅ S,.0.6H₂ O: C,56.90; H,7.86; N,9.05%); ν_(max) (Nujol) 3340, 1650 and 1625 cm⁻¹ ; δ (d⁶ DMSO) 0.82(6H,t,J=7 Hz, (CH₃)₂ CH), 1.17 and 1.5-1.9 (5H, m, CHCH(CH₃)₂ and SCH₂CH₂), 2.04 (3H, s,CH₃ S), 2.3 (2H, m, --SCH₂), 2.6 (3H, d, J=5 Hz,NHCH₃), 2.6-2.95 (3H, m, CH₂ C₆ H₄ and α-CH),), 3.14 (1H, t, J=7 Hz,α-CH), 3.7 (3H, s, OCH₃), 4.25 (1H, m, --CH), 6.8 and 7.12 (2×2H, eachd, each J=9 Hz, C₆ H₄), 7.88 (1H, q, J=5 Hz, NHCH₃) and 8.18 (1H, d, J=9Hz, NHCH); m/e 454 (100%, [m+1]⁺).

O-Methyl-L-tyrosine N-methylamide used in stage (a) above was preparedfrom Z-L-tyrosine as follows:

(i) Z-L-Tyrosine-O-methyl ether

Z-L-Tyrosine (150 g, 0.476M) was dissolved with stirring in diluteaqueous sodium hydroxide (42 g, 1.05M in 750 ml H₂ O). Dimethyl sulphate(51 ml, 0.54M) was then added dropwise over 30 min. to this solution atroom temperature. After 2 h further NaOH was added (4.2 g, 0.105M in 40ml H₂ O) followed by dimethyl sulphate (5.1 ml) after which the reactionwas allowed to stir overnight at room temperature. The reaction was thenacidified to pH 2, extracted with CH₂ Cl₂ and the CH₂ Cl₂ layer washedwith aqueous sodium chloride, dried (MgSO₄) and concentrated in vacuo toyield the crude product. Recrystallisation from ethyl acetate/hexanegave the required methyl ether (155 g); m.p. 114°-115°; (Found: C,65.84; H, 5.82; N, 4.22. C₁₈ H₁₉ NO₅ requires C, 65.64; H, 5.81; N,4.25%); ν_(max) (CHCl₃) 3412 and 1715 cm⁻¹ ; δ(CDCl₃) 3.1 (2H, m, CH₂ C₆H₄); 3.76 (3H, s, OCH₃); 4.66 (1H, dd, J=8 and 3 Hz, α-CH); 5.1 (2H, m,CH₂ C₆ H₅); 5.23 (1H, d, J=8 Hz, NH); 6.8 (2H, d, J=8.6 Hz, Tyr H-3,H-5); 7.05 (2H, d, J=8.6 Hz, Tyr H-2, H-6); 7.33 (5H, broad s, C₆ H₅);m/e 330 (68% [M+1]⁺), 285 (100% [M-CO₂ H]⁺).

(ii) N-(Benzyloxycarbonyl)-O-methyl-L-tyrosine N-Methylamide

To a stirred solution of N-(Benzyloxycarbonyl)-O-methyl-L-tyrosine (155g, 0.471M) in dry CH₂ Cl₂ was added 1-hydroxybenzotriazole (63.6 g,0.471M) followed by a solution of DCC (97.2 g, 0.471M) in CH₂ Cl₂ (100ml) added slowly at 0° C. After warming to room temperature over 1 hr, asolution of methylamine (30 g) in CH₂ Cl₂ (250 ml) was added dropwise tothe reaction mixture which was then stirred overnight at roomtemperature. The reaction was then filtered, washed with saturatedaqueous sodium bicarbonate (×2), dried (MgSO₄) and concentrated in vacuoto give a solid. Recrystallisation from ethyl acetate/hexane gave thedesired amide (142 g); m.p. 167°-170°; (Found: C, 66.72; H, 6.58; N,8.29. C₁₉ H₂₂ N₂ O₄ requires C, 66.65; H, 6.48; N, 8.18%) ν_(max)(CHCl₃) 3440, 1710 and 1672 cm⁻¹ ; δ(CDCl₃) 2.70 (3H, d, J=5 Hz, NCH₃);2.98 (2H, m, CH₂ C₆ H₄); 3.77 (3H, s, OCH₃); 4.30 (1H, dd, J=7.6 and 3Hz, α-CH); 5.06 (2H, m, OCH₂ C₆ H₅); 5.43 (1H, m, OCONH); 5.84 (1H, m,CONH); 6.80 (2H, d, J=8.6 Hz, Tyr H-3 and H-5); 7.15 (2H, d, J=8.6 Hz,Tyr H-2 and H-6); 7.32 (5H, m, C₆ H₅); m/e 343 (100%, [m+1]⁺).

(iii) O-Methyl-L-tyrosine N-Methylamide

To a solution of N-(Benzylocycarbonyl)-O-methyl-L-tyrosine N-methylamide(15.6 g, 0.056M) in ethanol (200 ml) and DMF (200 ml) was added 10% Pd/C(1 g) and trifluoroacetic acid (4 ml). Hydrogen was then passed throughthe solution for 3 h after which the reaction was filtered andconcentrated in vacuo. The residue was dissolved in H₂ O (150 ml),neutralised with sodium bicarbonate and extracted into CH₂ Cl₂ (150ml×5). The combined organic extracts were dried (Na₂ SO₄) andconcentrated in vacuo to yield an oil which subsequently crystallised.Recrystallisation from ethyl acetate/hexane gave O-methyl-L-tyrosineN-methylamide (9.0 g), m.p. 90°-91°; (Found: C, 63.49; H, 7.71; N, 13.44C₁₁ H₁₆ N₂ OA requires C, 63.44; H, 7.74; N, 13.45%) ν_(max) (CHCl₃)3350 and 1660 cm⁻¹ ; δ(CDCl₃) 1.3 (2H, br, NH ₂); 2.64 (1H, dd, J=13.8and 9.2 Hz, CHC₆ H₄); 2.80 (3H, d, J=5 Hz, NCH₃); 3.18 (1H, dd, J=13.8Hz and 4 Hz, CHC₆ H₄); 3.55 (1H, dd, J=9 Hz and 4 Hz, α-CH) 3.78 (3H, s,OCH₃); 6.85 (2H, d, J=8,2 Hz, Tyr H-3 and H-5); 7.12 (2H, d, J=8,2 Hz,Tyr H-2 and H-6); 7.28 (1H, br, CONH).

EXAMPLE 8N-[4-N-(benzyloxycarbonyl)amino-1-(R)-methoxycarbonylbutyl]-L-leucyl-O-methyl-L-tyrosineN-Methylamide

To a solution of methyl 5-N-(benzyloxycarbonyl)amino-2-bromo-pentanoate(10.3 g, 0.03M), L-leucyl-O-methyl-L-tyrosine N-methylamide (9.6 g,0.03M) and N-methyl morpholine in dry acetonitrile (100 ml) was addedsodium iodide (4.5 g, 0.03M). The mixture was then stirred and heatedunder reflux for 24 hr. The cooled reaction mixture was then filteredand evaporated in vacuo to yield an oil. Chromatography on silicaeluting with dichloromethane in an increasing ethyl acetate gradientgave the title compound (2.8 g); m.p. 124°-127°; (Found: C, 63.7; H,7.52; N, 9.56. C₃₁ H₄₄ N₄ O₇ requires C, 63.68; H, 7.58; N, 9.58%);ν_(max) (CHCl₃) 3400, 1718 and 1660 cm⁻¹ ; δ(CDCl₃) 0.85 and 0.87 (each3H, each d, each J=6 Hz, CH)CH₃)₂) 1.0-1.85 (8H, m, NHCH₂ CH₂, CH₂, CH₂CH and NH); 2.74 (3H, d, J=5 Hz, NCH.sub. 3); 2.96-3.42 (6H, m, NHCH₂,α-CH×2, CH₂ C₆ H₄); 3.66 (3H, s, OCH₃); 3.75 (3H, s, OCH₃); 4.6 (1H, dd,J=13 Hz and 6 Hz, α-CH); 5.0 (1H, m, OCONH); 5.1 (2H, s, CH₂ C₆ H₅);6.71 (1H, br, CONH); 6.80 (2H, d, J=8.6 Hz, Tyr H-3 and H-5); 7.10 (2H,d, J=8.6 Hz, Tyr H-2 and H-6); 7.35 (5H, m, C₆ H₅); 7.56 (1H, m, CONH);m/e 585 (100% [m+1]⁺).

The starting materials used in this preparation were synthesised asfollows:

(a) L-Leucyl-O-methyl-L-tyrosine N-methylamide

To a solution of BOC-L-Leucine (5.26 g, 0.021M) in CH₂ Cl₂ (40 ml) andDMF (10 ml) stirred at 0° was addedN-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (4 g,0.021M). After 15 min., N-methyl morpholine (0.021M) was added followedby, after a further 10 min. at 0°, a solution of O-methyl-L-tyrosineN-Methylamide (4.3 g, 0.019M) in CH₂ Cl₂. The reaction mixture wasallowed to warm to room temperature and stirred overnight. The reactionwas then concentrated in vacuo, and the residue in CH₂ Cl₂, washed inturn with H₂ O (200 ml), saturated aq. NaHCO₃ (200 ml), dilute HCl (1M;200 ml), saturated aq. NaHCO₃ (200 ml) and water (150 ml), dried (Na₂SO₄) and evaporated in vacuo to a solid. Recrystallisation from ethylacetate/hexane gaveN-(Tertiarybutoxycarbonyl)-L-leucyl-O-methyl-L-tyrosine N-methylamide asa white crystalline solid, (4.5 g), m.p. 159-161; (Found: C, 62.65; H,8.33; N, 9.96. C₂₂ H₃₅ N₃ O₅ requires C, 62.69; H, 8.37; N 9.97%).ν_(max) (CDCl₃) 3400, 1700 and 1662 cm⁻¹ ; δ(CDCl₃) 0.91 (6H, dd, J=7and 14 Hz, CH(CH₃)₂); 1.37 (9H, s, OC(CH₃)₃); 1.47-1.7 (3H, m, CH₂CH(CH₃)₂); 2.71 (3H, d, J=4.7 Hz, NHCH₃), 2.98 and 3.14 (each 1H, eachm, CH₂ C₆ H₄); 3.78 (3H, s, OCH₃); 4.0 and 4.61 (each 1H, each m,2×α-CH); 4.86, (1H, br s, OCONH); 6.40 and 6.55 (each 1H, each br s,CONH×2); 6.82 (2H, d, J=8.4 Hz, Tyr H-3 and H-5); 7.08 (2H, d, J=8.4 Hz,Tyr H-2 and H-6); m/e 422 (70%, [m+1]⁺), 365 (70%, [m-58]⁺).

To a stirred solution of

N-Tertiarybutoxycarbonyl)-L-leucyl-O-methyl-L-tyrosine N-methylamide(7.0 g, M) in CH₂ Cl₂ (40 ml) cooled at 10° was added trifluoroaceticacid (70 ml) and the resulting solution stirred at room temperature for1 h. The reaction was then concentrated in vacuo, and the residuedissolved in water, neutralised with sodium bicarbonate and extractedwith CH₂ Cl₂. The organic extracts were dried (Na₂ SO₄), filtered andconcentrated in vacuo to give L-leucyl-O-methyl-L-tyrosine N-methylamide(5.2 g); m.p. 128°-132°; (Found: C, 60.04; H, 8.72; N, 12.26; C₁₇ H₂₇ N₃O₃ requires C, 60.16; H, 8.61; N, 12.38%); ν_(max) (CDCl₃) 3325 and 1655cm⁻¹ ; [α]_(D) ²⁰ =10.2° (C=2.00, MeOH); δ(CD₃ OD) 0.88 and 0.92 (each3H, each d; 1.2-1.4 (1H, m, CH₂ CH(CH₃)₂); 1.44-1.8 (2H, m, CHCH(CH₃)₂);2.73 (3H, d, J=5 Hz, NCH₃); 2.82-3.3 (4H, m, NH₂, CH₂ C.sub. 6 H₄); 3.46(1H, m, CH); 3.76 (3H, s, OCH₃); 4.58 (1H, q, dd, J=8 and 3 Hz, α-CH);6.56 (1H, br, CONH); 6.82 (2H, d, J=8.6 Hz, Tyr H-3 and H-5); 7.13 (2H,d, J=8.6 Hz, Tyr H-2 and H-6); 7.96 (1H, d, J=8 Hz, CONH); m/e 322 (100%[m+1]⁺).

(b) Methyl 5-N-(Benzyloxycarbonyl)amino-2-bromopentanoate

To a stirred solution of ε-Z-ornithine (53.2 g, 0.1M) in dilute H₂ SO₄(2.5N, 500 ml) at 0° was added KBr (60 g, 0.5M). To this solution wasthen added portionwise sodium nitrite (7.6 g, 0.11M) whilst the reactiontemperature was maintained at 0° by the addition of ice. After stirringfor 1 h at 0° the reaction mixture was allowed to warm to roomtemperature over 2 h. Diethyl ether (500 ml) was then added and theaqueous layer was re-extracted with diethylether (500,; ×3). Thecombined ethereal extracts, were washed with water and then brine, dried(MgSO₄), filtered and concentrated to an oil in vacuo.

To the crude bromo-acid (45 g, 0.136M) in dry methanol (300 ml) cooledto -30° was added dropwise thionyl chloride (33.7 ml, 0.405M) at such arate that the temperature did not exceed -15°. The reaction mixture waswarmed to 10° over 2 h and stirred at room temperature for 30 min. andthen at 40° for 30 min. The resulting solution was then concentrated invacuo, dissolved in CH₂ Cl₂ and washed in turn with water, saturated aq.NaHCO₃ and water. The residue isolated from the organic layer waschromatographed on silica in 5% ethylacetate in CH₂ Cl₂ to give thetitle compound as an oil (10.3 g), (Found: C, 48.61; H, 5.61; N, 4.00.C₁₄ H₁₈ BrNO₄ requires C, 48.85; H, 5.27; N, 4.07%); δ(CDCl₃) 1.5-1.8and 1.9-2.2 (each 2H, each m, CH₂ CH₂), 3.23 (2H, q, J=6 Hz, NCH₂), 3.77(3H, s, OCH₃), 4.25 (1H, dd, J=7 and 14 Hz, α-CH), 4.8-4.9 (1H, broad s,NH), 5.10 (2H, s, OCH₂) and 7.35 (5H, broad s, C₆ H₅).

EXAMPLE 9N-[4-N-(Benzyloxycarbonyl)amino-1-(R)-carboxybutyl]-L-leucyl-O-methyl-L-tyrosineN-Methylamide

To a solution of the ester (650 mg, 1.14M) from Example 8 inmethanol/water (10:1, 11 ml) was added dilute NaOH (IN, 2.3 ml). Thereaction mixture was stirred for 6 h at room temperature, acidified withacetic acid and then concentrated to a semi-solid in vacuo. This waspartitioned between ethyl acetate and water and the resulting solid wasfiltered, washed with water and ethyl acetate and dried in vacuo to givethe title compound (585 mg); m.p. 164°-169°; (Found: C, 61.59; H, 7.24;N, 9.40. C₃₀ H₄₂ N₄ O₇ requires C, 61.21; H, 7.53; N, 9.52%); υ_(max)(Nujol) 3320, 1690 and 1645 cm⁻¹ ; δ(d⁶ DMSO) 0.85 (6H, m, CH(CH₃)₂);0.96-1.8 (7H, m, CH₂ CH(CH₃)₂, NHCH₂ CH₂ CH₂); 2.57 (3H, d, J=5 Hz,NCH₃); 2.5-3.2 (6H, m, NHCH₂, CH₂ C₆ H₄, α-CH×2); 3.70 (3H, s, OCH₃);4.42 (1H, m, α-CH); 5.0 (2H, s, CH₂ C₆ H₅); 6.78 (2H, d, J=8.6 Hz, TyrH-3 and H-5); 7.10 (2H, d, J=8.6 Hz, Tyr H-2 and H-6); 7.20 (1H, m,CONH); 7.35 (5H, m, C₆ H₅); 7.88 (1H, m, CONH); 8.18 (1H, m, CONH).

EXAMPLE 10 N-[4-N-[N-(Acetyl)-L-prolyl-L-leucyl]amino-1-(R)-carboxybutyl]-L-leucyl-O-methyl-L-tyrosine N-Methylamide

This was synthesised from Z-proline, leucine methyl ester andN-[4-N-(benzyloxycarbonyl)amino-1-(R)-methoxycarbonylbutyl]-L-leucyl-O-methyl-L-tyrosineN-methylamide as described in the following paragraphs:

(a) N-(Benzyloxycarbonyl)-L-prolyl-L-leucine Ethyl Ester

To a stirred solution of Z-L-proline (12.7 g, 0.051M) in CH₂ Cl₂ (200ml) cooled to 0° was added 1-hydroxy benzotriazole (7.0 g) followed by asolution of DCC (10.6 g) in CH₂ Cl₂ (50 ml). After 30 min. at 0°L-leucine ethyl ester (10.0 g, 0.051 molM) was added followed bytriethylamine (15 ml) and the reaction mixture was then left to stir andwarm up to room temperature overnight. The reaction mixture was thenfiltered and washed in turn with saturated aq. NaHCO₃ (250 ml×3), H₂ O(250 ml), dilute aq. HCl (1 M, 250 ml×3) and water (250 ml×2). Theorganic layer was dried (Na₂ SO₄), filtered and concentrated in vacuo toan oil which subsequently crystallised. Recrystallisation from ethylacetate/hexane gave N-(benzyloxycarbonyl)-L-prolyl-L-leucine ethyl esteras a white crystalline solid, 15.5 g, (78%); m.p. 67°-68°; (Found: C,64.55; H, 7.79; N, 7.22. C₂₁ H₃₀ N₂ O₅ requires C, 64.61; H, 7.74; N,7.17%); ν_(max) (CHCl₃) 1740 and 1680 cm⁻¹ ; δ(CDCl₃) 0.7-0.95 (6H, m,CH(CH₃)₂); 1.18 (3H, m, OCH₂ CH₃); 1.3-1.95 and 2.05-2.25 (7H, m, CH₂CH₂, CH₂ CH(CH₃)₂); 3.4 (2H, m, CH₂ N); 4.05 (2H, m, OCH₂ CH₃); 4.25(2H, m, α-CH); 4.98 and 5.05 (together 2H, respectively q, J=7 Hz, andm, CH₂ C₆ H₅); 7.35 (5H, broad s, C₆ H₅) and 8.26 (1H, m, CONH); m/e 391(100%, [m+1]⁺).

(b) N-Acetyl-L-proline-L-leucine Ethyl Ester

To a solution of N-(benzyloxycarbonyl)-L-prolyl-L-leucine ethyl ester(7.5 g, 0.02 mM) in methanol (100 ml) was added acetic acid and 10% Pd/C(0.8 g). After stirring under hydrogen for 3 h at room temperature thereaction was filtered and concentrated to an oil in vacuo. Trituation ofthe residue with ether and recrystallisation from ethyl acetate/hexanegave L-prolyl-L-leucyl ethyl ester as the acetate salt (5.0 g), m.p.87°-89°. ν_(max) 1760 and 1660 cm⁻¹ ; δ(CDCl₃) 0.94 (6H, m, CH(CH₃)₂);1.27 (3H, t, J=7 Hz, OCH₂ CH₃); 1.45-2.35 (7H, m, CH₂ CH₂, CH₂CH(CH₃)₂); 2.2 (3H, s, CH₃ CO₂); 3.1 (2H, m, CH₂ N); 4.15 (1H, m, α-CH);4.19 (2H, q, J=7 Hz, OCH₂ CH₃); 4.55 (1H, m, α-CH); 7.24 (2H, br, NH,CO₂ H); 7.87 (1H, d, J=7 Hz, CONH); m/e (100% [m+1]⁺)].

To a solution of the foregoing amine (3.0 g, 11.7 mM) in CH₂ Cl₂ (50 ml)was added p-nitrophenylacetate (2 g, 12MM). After stirring the reactionmixture at room temperature for 3 days it was diluted with CH₂ Cl₂ (350ml), washed with water, dried (Na₂ SO₄) and concentrated to an oil invacuo. Chromatography on silica in 1:1 CH₂ Cl₂ /EtOAc followed by 9:1CH₂ Cl₂ /MeOH yielded N-acetyl-L-prolyl-L-leucine ethyl ester as a paleyellow oil (2.2 g); (Found [m+1]⁺ =299.19704. C₁₅ H₂₇ N₂ O₄ requires[m+1]⁺ =299.19707); γ_(max) (CHCl₃) 3600-3100 (broad), 1735, 1675 and1625 cm⁻¹ ; δ(CDCl₃) 0.95 (6H, m, CH(CH₃)₂); 1.25 (3H, t, J=7 Hz, OCH₂CH ₃); 1.44-2.5 (7H, m, CH₂ CH₂, CH₂ CH(CH₃)₂); 2.12 (3H, s, CH₃ CO);3.36-3.7 (2H, m, CH₂ N); 4.18 (2H, t, J=7 Hz, OCH₂ CH₃); 4.25-4.55 (1H,m, CH Pro); 4.6 (1H, CH Leu); 6.38 and 7.35 (1H, each d, J=7 Hz, CONH).

(c)N-[4-N-[N-(Acetyl)-L-prolyl-L-leucyl]amino-1-(R)-methoxycarbonylbutyl]-L-leucyl-O-methyl-L-tyrosineN-methylamide

To a solution ofN-[4-N-(benzyloxycarbonyl)amino-1-(R)-methoxycarbonylbutyl]-L-leucyl-O-methyl-L-tyrosineN-Methylamide (570 mg, 0.97 mM) in methanol (8 ml) was added 10% Pd/Cand dilute HCl (1M, 2 ml). After stirring the reaction mixture underhydrogen for 2 h at room temperature it was filtered and concentrated invacuo to a solid, (490 mg) (100%), which was used as such in the nextstep.

N-Acetyl-L-prolyl-L-leucine (271 mg, 1.06MM; obtained from the foregoingethyl ester by hydrolysis in methanol with one equivalent of IN-sodiumhydroxide solution at 20° over 16 h followed by neutralisation withdilute HCl) in CH₂ Cl₂ (2 ml) and DMF (2 ml) was stirred at 0° and1-hydroxy benzotriazole (162 mg, 1.06 mM) andN-ethyl-N'-(dimethylaminopropyl)carbodiimide hydrochloride (240 mg, 1.06mM) were then added. After 5 min N-methylmorphine (107 mg, 1.06 mM) wasadded followed, after 15 min, by the amine hydrochloride (preparedabove) (485 mg, 0.96 mM). After stirring overnight at 0°-4°, thereaction mixture was concentrated in vacuo, dissolved in CH₂ Cl₂ andwashed in turn with water, saturated aq. NaHCO₃ and dilute HCl. The acidlayer was separated, neutralised with NaHCO₃ and extracted with CH₂ Cl₂.The organic extracts were dried, (Na₂ SO₄) and evaporated in vacuo toyield the title compound as a foam (570 mg); m.p. 68°-72°; (Found: C,59.99; H, 8.35; N, 11.65. C₃₆ H₅₆ N₆ O₈.1H₂ O requires C, 59.98; H, ;b8.39; N, 11.66;l %); δ(d⁶ DMSO) 0.82 (12H, m, CH(CH₃)₂ ×2); 1.0-2.34(14H, m, CH₂ CH₂ ×2, CH₂ CH(CH₃)₂ ×2); 1.98 and 2.0 (together 3H, eachs, CH₃ CO); 2.50-3.08 (8H, m, CH₂ C₆ H₄, CH₂ N×2 and 2×α-CH); 2.56 (3H,d, J=5 Hz, CH₃ N); 3.54 (3H, s, OCH₃); 3.70 (3H, s, OCH₃); 4.0-4.5 (3H,m, α-CH); 6.78 (2H, d, J=8 Hz Tyr H-3 and H-5); 7.11 (2H, d, J=8.6 Hz,Tyr H-2 and H-6); 7.5-8.35 (4H, m, CONH).

(d)N-[4-N-[N-(Acetyl)-L-prolyl-L-leucyl]amino-1-(R)-carboxybutyl]-L-leucyl-O-methyl-L-tyrosineN-Methylamide

To a solution of the preceeding ester (380 mg, 0.54 mM) in methanol (5ml) was added dilute NaOH (1M, 1 ml). After stirring overnight at roomtemperature, the reaction mixture was neutralised with acetic acid andconcentrated in vacuo. Chromatography on reverse phase silica in 1:1MeOH/H₂ O gave the title compound (280 mg); m.p. 97°-101°; (Found: C,58.52; H, 7.93; N, 11.46. C₃₆ H₅₆ N₆ O₈.1.5H₂ O requires C, 58.72; H,8.31; N, 11.74%). ν_(max) (Nujol) 3700-3140 (broad) and 1635 cm⁻¹ ;δ(CD₃ OD) 0.9 (12H, M, 2×CH(CH₃)₂); 1.4-2.25 (14H, M, 2×CH₂ CH₂, 2×CH₂CH(CH₃)₂); 1.98 and 2.0 (together 3H, each s, CH₃ CO); 2.68 and 2.72(together 3H, each s, CH₃ N), 2.75-3.8 (8H, m, CH₂ C₆ H₅, CH₂ N×2,2×CH); 3.75 (3H, s, OCH₃); 4.25-4.65 (3H, m, αCH), 6.78 (2H, d, J=8.6Hz, Tyr H-3 and H-5); 7.11 (2H, d, J=8.6 Hz, Tyr H-2 and H-6).

EXAMPLE 11N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypropyl]-L-leucyl-O-methyl-L-tyrosineN-Methylamide

This was prepared in two stages from methyl4-N-(benzyloxycarbonyl)amino-2-bromo-butanoate andL-leucyl-O-methyl-L-tyrosine N-methylamide as described below:

(a) N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosine N-Methylamide

Methyl 4-N-(benzyloxycarbonyl)amino-2-bromobutanoate (30 g),L-leucyl-O-methyl-L-tyrosine N-methylamide (30 g) and N-methylmorpholine (9.4 g) in acetonitrile (250 ml) was stirred and heated underreflux overnight. A further portion of the amine (1.1 g) was then addedand the solution was heated under reflux for a further 4 h. The reactionmixture was then concentrated in vacuo, dissolved in chloroform and thesolution washed with saturated aq. sodium bicarbonate solution. Thematerial isolated from the organic layer was chromatographed on silicawith ethyl acetate as eluant to yieldN-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosineN-methylamide (11.7 g); (Found: C, 63.09; H, 7.46; N, 9.59. C₃₀ H₄₂ N₄O₇ requires C, 63.16; H, 7.37; N. 9.83%); ν_(max) (CHCl₃) 3400, 1720 and1660 cm⁻¹ ; δ(CDCl₃) 0.86 (6H, m, CH(CH₃)₂); 1.2-2.1 (6H, m, NHCH₂ CH₂CH, CH₂ CH(CH₃)₂, NH); 2.77 (3H, d, J=5 Hz, NCH₃); 2.95-3.45 (5H, m,NHCH₂, CH₂ C₆ H₄, αCH); 3.66 and 3.76 (each 3H, each s, 2×OCH₃); 3.8 and4.61 (each 1H, each m, 2×CH); 5.10 (2H, m, CH₂ C₆ H₅); 5.21 (1H, m,OCONH); 672 (1H, m, CONH); 6.81 (2H, d, J=8.6 Hz, Tyr H-3 and H-5); 7.12(2H, d, J=8.6 Hz, Tyr H-2 and H-6); 7.35 (5H, s, C₆ H₅); 7.55 (1H, d,J=8 Hz, CONH); m/e 571 (100% [m+1]⁺).

The methyl 4-N-(benzyloxycarbonyl)amino-2-bromobutanoate required inthis preparation was made from L-glutamic acid as described in thefollowing paragraphs:

L-Glutamic acid (105 g, 0.713M) was dissolved in concentrated sulphuricacid (300 ml) and to this was added chloroform (300 ml). To the stirredbi-phasic mixture at 0° was added portionwise over 30 min. sodium azide(60 g, 0.9 mole). The reaction mixture was stirred at 5°-10° for 30 min.and was then allowed to slowly warm to room temperature. The reactionmixture was then slowly heated to 80° for one hour the reaction wascooled, poured into water (1.5 l) and the aqueous layer was separated.The aqueous extract was diluted (to 20 liters) and was then applied toDowex 50WX8, 16-40 mesh, H⁺ resin. The column was washed with water andthen with 1:1 880 Ammonia/Water and the fractions containing the productwere lyophilised.

The crude product obtained above was dissolved in water (1 liter) and tothis was added basic copper carbonate (100 g). The stirred mixture washeated under reflux for 40 min. and the hot solution was filtered. Thesolution was cooled to 35° and NaHCO₃ (60 g) and CHCl₃ (300 ml) wereadded. After stirring for 30 min. at room temperature, benzyloxycarbonylchloride (75 ml) was added and the mixture was then allowed to stir atroom temperature overnight. A further portion of benzyloxycarbonylchloride (30 ml) was then added and stirring was continued for a further24 h. The crystalline copper complex which had precipitated was thenfiltered, washed with water and added to a solution of EDTA (di Na salt)(120 g) in water (1.5 liter). The resulting mixture was stirred andheated under reflux for 3 h and was then cooled to 5°. After 40 h at 5°the crystalline product was collected by filtration, washed with waterand acetone and dried in vacuo at 45°.

The 4-Z-diamino-butyric acid from above (120 g) was suspended in amixture of dilute sulphuric acid (1M, 600 ml), water (200 ml) andpotassium bromide (240 g). Sufficient water (200 ml) was then added toform a single phase. To the resulting solution stirred at -7° to -9°,was added a solution of sodium nitrite (44 g) in H₂ O dropwise over 1 h.After 30 min at -7°, the mixture was warmed to room temperature over 1h. Diethyl ether (1.5 liters) was added and the separated aqueous layerwas washed with a further portion of ether. The dried ethereal extractswere concentrated in vacuo and the residue in methanol (1 liter) wascooled to 0° and treated dropwise with thionyl chloride (65 ml). Thereaction was then concentrated in vacuo and the residue was partitionedbetween diethyl ether and saturated aq. sodium bicarbonate. The materialisolated from the ether was chromatographed on silica eluting with agradient of ethyl acetate in hexane to give methyl4-N-(benzyloxycarbonyl)amino-2-bromo-butanoate (90 g) as an oil whichcrystallised on standing, m.p. 46°-50°; (Found: C,47.17; H,5.01; N,4.16.C₁₃ H₁₆ BrNO₄ requires C,47.29; H,4.88; N,4.24%); δ (CDCl₃) 2.08-2.45(2H,m,CH₂); 3.37 (2H,m,NHCH₂); 3.76 (3H,s,OCH₃); 4.32 (1H,dd,J=10 Hz and6 Hz, CH); 4.97 (1H,broad s, OCONH); 5.09 (2H,s,OCH₂) and 7.34 (5H,s,C₆H₅).

(b)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypropyl]-L-leucyl-O-methyl-L-tyrosineN-Methylamide

To a solution of the preceeding ester (171 mg, 0.3 mM) in methanol (10ml) stirred at 0° was added dilute NaOH (1N, 0.6 ml). After stirringovernight at 0° a further portion of NaOH (1N, 0.3 ml) was added and thesolution was then stirred for 6 h at room temperature. The reactionmixture was then acidified with acetic acid and concentrated to a solidin vacuo. Recrystallisation of this material from methanol/H₂ O gave thetitle compound (150 mg); m.p. 170°-172°; (Found: C,60.97; H,7.11;N,9.68. C₂₉ H₄₀ N₄ O₇ +0.8H₂ O requires C,60.99; H,7.34; N,9.81%);ν_(max) (Nujol) 3330, 1690 and 1640 cm⁻¹ ; δ(CD₃ OD) 0.88 (6H,dd,J=14 Hzand 7 Hz, CH(CH₃)₂); 1.2-1.95 (5H,m,NHCH₂ CH₂, CH₂ CH(CH₃)₂); 2.69(3H,s,NCH₃); 2.75-3.65 (6H,m,NHCH₂, CH₂ C₆ H₄, and α-CH×2); 3.74(3H,s,OCH₃); 4.54 (1H,dd,J=10 Hz and 6 Hz, α-CH); 5.08 (2H,m,CH₂ C₆ H₅);6.82 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.12 (2H,d,J=8.6 Hz, Tyr H-2 andH-6); 7.35 (5H,m,C₆ H₅).

EXAMPLE 12N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosineN-Methylamide

To a solution of N-(Tertiarybutoxycarbonyl)-L-leucyl-O-methyl-L-tyrosylN-methylamide (4.2 g, 0.01M) in CH₂ Cl₂ (5 ml) at 18° was addedtrifluoroacetic acid (8 ml). After stirring for 2 h at room temperaturethe reaction was concentrated in vacuo and was then trituated with dryether to yield a gum. This was taken up in methanol (25 ml), methyl4-N-(benzyloxycarbonyl)amino-2-oxo-butanoate (4.0 g; 0.015M; Synthesis,(1982), 41) was added and the pH of the solution adjusted to 6.5 withtriethylamine. To this solution stirred at 0° was added sodiumcyanoborohydride (400 mg) portionwise whilst the pH was periodicallyre-adjusted to 6.5 by the addition of acetic acid. After 1 h furthersodium cyanoborohydride (400 mg) was added and the reaction was stirredovernight at room temperature. After concentration in vacuo the residuewas partitioned between CH₂ Cl₂ (100 ml) and water (50 ml). The CH₂ Cl₂layer was separated, washed in turn with dilute HCl (1M, 20 ml), water(25 ml), saturated sodium bicarbonate solution (2×30 ml), dried andevaporated to an oil. Chromatography on silica in CH₂ Cl₂ in anincreasing ethyl acetate gradient gave the title compound as a foam (1.0g) which had physical data identical to that given above in Example 11.

EXAMPLE 13 N-[3-Amino-1-(R)-carboxypropyl]-L-leucyl-O-methyl-L-tryosineN-Methylamide

The acid (320 mg, 0.56 mM) from Example 11 in methanol (10 ml) wastreated with dilute HCl (1M, 1 ml). This solution was hydrogenated over10% palladium on charcoal (60 mg) for 90 min. at room temperature,filtered and then concentrated in vacuo to give the title compound asits dihydrochloride salt; m.p. 149°-152° (from CH₂ Cl₂ -ether); (Found:C,48.17; H,6.98; N,10.49. C₂₁ H₃₄ N₄ O₅.2HCl+0.5 CH₂ Cl₂ requiresC,48.01; H,6.93; N,10.42%); ν_(max) (Nujol) 3650-2400 (br), 1730 and1650 cm⁻¹ ; δ(CD₃ OD) 0.92 and 0.95 (each 3H, each d, each J=15Hz,CH(CH₃)₂); 1.45-1.90 (3H,m,CH₂ CH(CH₃)₂); 2.25 (2H,m,NHCH₂ CH₂); 2.68(3H,s,OCH₃); 3.04 (4H,m,NHCH₂ and CH₂ C₆ H₄); 3.58 (1H,dd,J=8 Hz and 6Hz,α-CH); 3.77 (3H,s,OCH₃); 3.94 (1H,dd,J=8 Hz and 4 Hz,α-CH); 4.64(1H,dd,J=13 Hz and 6 Hz,α-CH); 6.88 (2H,d,J=8.6 Hz, Tyr H-3 and H-5) and7.10 (2H,d,J=8.6 Hz, Tyr H-2 and H-6).

EXAMPLE 14N-[3-N-(p-Nitrobenzyloxycarbonyl)amino-1-(R)-carboxypropyl]-L-leucyl-O-methyl-L-tyrosineN-Methylamide

(a)N-[3-N-(p-Nitrobenzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosineN-Methylamide

A solution of N-[3-N-(benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosine N-methylamide (1.24 g, mM) inmethanol (25 ml) containing ethereal HCl (1 ml of a 2.6M solution) washydrogenated over 10% palladised charcoal (0.3 g) for 6 h at 20°. Thesolution was filtered and concentrated in vacuo to giveN-[3-N-amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosineN-methylamide dihydrochloride as a foam (1.2 g) which was used in thenext step without further purification.

To a suspension ofN-[3-N-amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosineN-methylamide dihydrochloride (400 mg, 0.808 mM) in dry CH₂ Cl₂ (6 ml)cooled in an ice bath, was added p-nitrobenzyloxycarbonyl chloride (400mg) in dry CH₂ Cl₂. To this was then added dropwise a solution ofN-methyl morpholine (270 mg, 2.67 mM) in dry CH₂ Cl₂ (2 ml). After 30min at 0°, a further portion of p-nitrobenzyloxycarbonyl chloride (400mg) in dry CH₂ Cl₂ (1 ml) was added followed by a solution of NMM (100mg) in dry CH₂ Cl₂ (1 ml). After a further 0.5 h at 0° the reactionmixture was diluted with CH₂ Cl₂ (20 ml), washed in turn with water (20ml), aq. citric acid solution (20 ml) and saturated aq. NaHCO₃ -(20 ml).The organic extract was concentrated in vacuo and purified bychromatography on silica eluting with CH₂ Cl₂ in a rapidly increasingethyl acetate gradient to give the title compound as a foam (450 mg,90%); (Found: [m+1]⁺ =616.3012. C₃₀ H₄₂ N₅ O₉ requires [m+1]⁺=616.2983); _(max) (CHCl₃) 3380, 1742 and 1660 cm⁻¹ ; m/e 616 (5%[m+1]⁺); 153 (100% [O₂ NC₆ H₄ CH₂ OH]⁺). δ(CDCl₃ 0.87 (6H,m,CH(CH₃)₂);1.1-2.0 (5H,m,NHCH₂ CH₂, CH₂ CH(CH₃)₂,NH) 2.76 (3H,d,J=5 Hz,NCH₃);2.9-3.5 (6H,m,NHCH₂,CH₂ C₆ H₄,α-CH×2) 3.68 and 3.77 (each 3H, each s,2×OCH₃); 4.60 (1H,dd,J=13 Hz and 6 Hz,α-CH); 5.10 (2H,s,CH₂ C₆ H₄ NO₂);5.45 (1H,m,OCONH); 6.50 (1H, broad s, OCONH); 6.82 (2H,d,J=8.6 Hz, TyrH-3 and H-5); 7.11 (2H,d,J=8.6 Hz, Tyr H-2 and H-6); 7.45 (1H,d,J=8 Hz,CONH); 7.52 (2H,d,J=9 Hz, benzoyl H-2 and H-6); 8.21 (2H,d, J=9 Hz,benzoyl H-3 and H-5).

(b)N-[3-N-(p-Nitrobenzyloxycarbonyl)amino-1-(R)-carboxypropyl]-L-leucyl-O-methyl-L-tyrosineN-methylamide

To a solution of the preceeding ester (360 mg, 0.58 mM) in methanol (6ml) at 0° was added dilute NaOH (1N, 1.2 ml). After standing at 0° for48 h, the solution was acidified with acetic acid and concentrated to asolid in vacuo. Trituration with ethyl acetate and water gave the titlecompound (56 mg); m.p. 167°-170°; (Found: C,56.56; H,6.58; N,11.21. C₂₉H₃₉ N₅ O₉ +0.8H₂ O requires C,56.54; H,6.64; N,11.37%); ν_(max) (Nujol)3250, 1690 and 1642 cm⁻¹ ; δ(d⁶ DMSO) 0.8 (6H,m,CH(CH₃)₂ ; 1.1-2.0(5H,m,NHCH₂ CH₂, CH₂ CH(CH₃)₂); 2.57 (3H,d,J=5 Hz,NCH₃); 2.62-3.85(7H,m,NCH₂, α-CH×2, CH₂ C₆ H₄, OH); 3.67 (3H,s,OCH₃); 4.43 (1H,m,α-CH);5.10 (2H,s,OCH₂); 6.78 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.13(2H,d,J=8.6 Hz, Tyr H-2 and H-6); 7.95 (1H,m,CONH); 8.07 (2H,d,J=8.6 Hz,Benzoyl H-2 and H-6); 8.25 (1H,m,CONH); 8.31 (2H,d,J=8.6 Hz, Benzoyl H-3and H-5); 9.12 (1H,m,CONH).

EXAMPLE 15N-[3-N-(Benzoyl)amino-1-(R)-carboxy-propyl]-L-leucyl-L-tyrosineN-Methylamide

This was prepared in two steps fromN-[3-N-amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosineN-methylamide as described below:

(a)N-[3-N-(Benzoyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-L-tyrosineN-Methylamide

To a stirred suspension ofN-[3-N-amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosineN-methylamide dihydrochloride (539 mg, 1 mM) and benzoyl chloride (186mg, 1 mM) in dry CH₂ Cl₂ (30 ml) at 0° was added dropwise N-methylmorphline (439 mg, 4.3 mM). The reaction mixture was then stirredovernight, concentrated in vacuo and chromatographed on silica elutingwith ethyl acetate in an ethyl acetate/methanol gradient to yield thetitle compound (350 mg); m.p. 145°-148°; (Found: C,63.97; H,7.38;N,10.20. C₂₉ H₄₀ N₄ O₆ +0.2H₂ O requires C,64.00; H,7.48; N,10.29%).δ(CDCl₃) 0.85 and 0.86 (each 3H, each d, each J=6.5 Hz, CH(CH₃)₂);1.18-1.80 (4H,m,NHCH₂ CH₂ CH and CH₂ CH(CH₃)₂,NH); 2.0 (2H,dd,J=13 and 6Hz, CH ₂ CH(CH₃)₂); 2.75 (3H,d,J=5 Hz,NCH₃); 3.06 and 3.4-3.7(6H,m,NHCH₂, CH₂ C₆ H₄ and α-CH×2); 3.64 and 3.74 (each 3H, each s,2×OCH₃) 4.60 (1H,dd,J=15 Hz and 6 Hz,α-CH); 6.5 and 6.75 (each 1H, eachm, 2×CONH); 6.82 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.15 (2H,d,J=8.6 Hz,Tyr H-2 and H-6); 7.5 (5H,m,C₆ H₄) and 7.77 (1H,d,J=8 Hz,CONH).

(b) N-[3-N-(Benzoyl)amino-1-(R)-carboxypropyl]-L-leucyl-L-tyrosineN-Methylamide

To the preceeding ester (150 mg, 0.27 mM) in methanol (10 ml) was addeddilute NaOH (1N, 1 ml) and the solution was then stirred at roomtemperature for 3 days. The reaction mixture was acidified with aceticacid and was concentrated in vacuo. Recrystallisation of the residuefrom methanol-H₂ O gave the title compound (110 mg); m.p. 175°-177°;(Found: C,61.41; H,7.71; N,10.17. C₂₈ H₃₈ N₄ O₆ +1.2H₂ O requiresC,61.34; H,7.34; N,10.22%); νmax (Nujol) 3320 and 1640 cm⁻¹ ; δ(d⁶ DMSO)0.82 (6H,m,CH(CH₃)₂); 1.05-2.0 (5H,m,NHCH₂ CH₂,CH(CH₃)₂); 2.58 (3H,d,J=5Hz,NCH₃); 3.65-4.55 (6H,m,NHCH₂),CH₂ C₆ H₄ and α-CH×2);3.68 (3H,s,OCH₃); 4.42 (1H,m,α-CH); 6.78 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.11(2H,d,J=8.6 Hz, Tyr H-2 and H-6); 7.46 (3H,m,CONH and 2 protons from C₆H₅); 7.86 (3H,m, 3 protons from C₆ H₅); 8.20 (2H,d,J=8 Hz,CONH); 8.51(1H,m,CONH).

EXAMPLE 16N-[3-N-(P-Nitrobenzoyl)amino-1-(R)-carboxypropyl]-L-leucyl-O-methyl-L-tyrosine.N-Methylamide

This was prepared exactly as described for the N-benzoyl derivative inExample 15 except that p-nitrobenzoyl chloride was used in place ofbenzoyl chloride in the first step. After hydrolysis of the intermediateester, the resulting solid was recrystallised from methanol-water togive the title compound, (450 mg); m.p. 170°-180°; (Found: C,57.38;H,6.82; N,11.86. C₂₈ H₃₇ N₅ O₈ +0.8H₂ O requires C,57.39; H,6.64;N,11.95%; ν_(max) (Nujol) 3340 and 1645 cm⁻¹ ; δ(d⁶ DMSO) 0.82(6H,m,CH(CH₃)₂ ; 1.05-2.05 (5H,m,NCH₂ CH₂ CH, CH₂ CH(CH₃)₂); 2.58(3H,m,NCH₃); 2.6-3.65 (6H,m,NHCH₂ α-CH×2 and CH₂ C₆ H₄); 3.7(3H,m,OCH₃); 4.45 (1H,m,α-CH); 6.8 (2H,d,J=8.6 Hz, Tyr H-3 and H-5);7.12 (2H,d,J=8.6 Hz, Tyr H-2 and H-6); 7.88 (1H,m,CONH); 8.08 (2H,d,J=8Hz, Benzoyl H-2 and H-6); 8.2 (1H,d,J=8 Hz,CONH); 8.33 (2H,d,J=8 Hz,Benzoyl H-3 and H-5) and 8.88 (1H,m,CONH).

EXAMPLE 17N-[3-N-(p-Aminobenzoyl)amino-1-(R)-carboxypropyl]-L-leucyl-O-methyl-L-tyrosineN-Methylamide

The acid (351 mg), from Example 16 was dissolved in methanol (25 ml) andto this solution was added 10% Pd/C (400 mg) and dilute ethereal HCl(2.6M, 2 ml). after stirring the reaction mixture under hydrogen for 2.5h at room temperature it was filtered and concentrated in vacuo to yieldthe title compound as a foam (290 mg);

m.p. 155°-160°; (Found: C,50.39; H,6.68; N,10.23. C₂₈ H₃₉ N₅ O₆ 3HCl+1H₂O requires C,50.26; H,6.62; N,10.46%); ν_(max) (Nujol) 3650-2120(broad), 1730 and 1645 cm⁻¹ ; δ (d⁶ DMSO) 0.81 and 0.87 (each 3H, eachs, CH(CH₃)₂); 1.3-1.8 (3H,m,CH(CH₃)₂); 2.05 (2H,m,NHCH₂ CH₂ CH); 2.58(3H,d,NCH₃); 2.75 and 2.98 (together 2H, each m, CH₂ C₆ H₄); 3.2-3.5(3H,m,NHCH₂ and α-CH); 3.7 (3H,s,OCH₃); 3.97 (1H,m,α-CH); 4.58(1H,m,α-CH); 6.83 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.01 (2H,d,J=8 Hz,benzoyl H-3 and H-5); 7.10 (2H,d,J-6.8 Hz, Tyr H-2 and H-6); 7.81(2H,d,J=8 Hz, benzoyl H-2 and H-6); 8.17 (1H,m,CONH); 8.67 (1H,m,CONH);9.11 (1H,d,J=8 Hz,CONH) and 9.5 (3H,br,NH₃).

EXAMPLE 18N-[3-(N'-Benzyl)carbamoyl-1-(R)-carboxypropyl]-L-leucyl-O-methyl-L-tyrosineN-Methylamide

This was prepared according to the following steps:

(a)N-[3-(N'-Benzyl)carbamoyl-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosineN-Methylamide

To a stirred suspension ofN-[3-N-amino-1-(R)-methoxy-carbonylpropyl]-L-leucyl-O-methyl-L-tyrosylN-Methylamide dihydrochloride (406 mg, 0.78 mM) in dry CH₂ Cl₂ (10 ml)at 0° was added benzyl isocyanate (104 μl, 1.56 mM). A solution ofN-methyl morpholine (189 mg, 1.87 mM) in dry CH₂ Cl₂ (5 ml) was thenadded dropwise over 5 min. After 30 min at 0°, a further portion ofbenzyl isocyanate (25 μl) was added and this was repeated after anadditional 30 min. at 0°. The reaction mixture was then allowed to warmto room temperature over 3 h. Water (50 ml) and CH₂ Cl₂ (50 ml) werethen added and the material isolated from the organic extracts waschromatographed on silica in 5% MeOH in CH₂ Cl₂ to afford the titlecompound (223 mg); 61°-69°; (Found: C,62.77; H,7.64; N,12.03. C₃₀ H₄₃ N₅O₆ +0.3H₂ O requires C,62.65; H,7.64; N,12.08%); δ(CDCl₃) 0.87(6H,m,CH(CH₃)₂) 1.10-2.0 (6H,m,NHCH₂ CH₂,CH₂ CH(CH₃)₂ and NH) 2.64(3H,d,J=5 Hz,NCH₃); 2.85-3.54 (6H,m,NHCH₂, CH₂ C₆ H₄ and α-CH×2); 3.67and 3.78 (each 3H, each s, 2×OCH₃); 4.37 (2H,dd,15 Hz and 2 Hz,CH₂ C₆H₅); 4.56 (1H,dd,J=13 Hz and 6 Hz,α-CH); 5.16, 5.42 and 6.44 (each 1H,each broad s, 3×CONH) 6.80 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.08(2H,d,J=8.6 Hz, Tyr H-2 and H-6); 7.3 (5H,m,C₆ H₅) and 7.7 (1H,d,J=8Hz,CONH).

(b)N-[3-(N'-Benzyl)carbamoyl-1-(R)-carboxypropyl]-L-leucyl-O-methyl-L-tyrosineN-Methylamide

To a solution of the preceeding ester (240 mg, 0.42 mM) in methanol (25ml) at room temperature, was added dilute NaOH (1N, 1.5 ml). Afterstanding overnight at room temperature the reaction mixture wasacidified with acetic acid and concentrated in vacuo. Chromatography onreverse phase silica eluting with a methanol/H₂ O gradient gave thetitle compound (107 mg); m.p. 104°-108°; (Found: C,60.88; H,7.44;N,12.12. C₂₉ H₄₁ N₅ O₆ H₂ O requires C,60.71; H,7.55; N,12.20%); ν_(max)(Nujol) 3300 and 1640 cm⁻¹ ; δ(d⁶ DMSO) 0.8 (6H,m,CH(CH₃)₂) 0.95-1.85(5H,m,NHCH₂ CH₂ and CH₂ CH(CH₃)₂); 2.2-3.4 (6H,m,NHCH₂, α-CH×2 and CH₂C₆ H₄); 2.56 (3H,d,J=5 Hz, NHCH₃); 3.70 (3H,s,OCH₃); 4.22 (2H,m,CH₂ C₆H₅); 4.45 (1H,m,α-CH); 6.0 and 6.42 (each 1H, each m, 2×CONH); 6.82(2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.12 (2H,d,J=8.6 Hz, Tyr H-2 and H-6);7.28 (5H,m,C₆ H₅); 7.94 (1H,m,CONH) and 8.25 (1H,d,J=Hz,CONH).

EXAMPLE 19N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypropyl]-L-leucineN-Phenethylamide

N-(Tertiarybutoxycarbonyl)-L-leucine (10 g, 0.04M in a mixture of CH₂Cl₂ (100 ml) and DMF (10 ml) was cooled to 0°. to this was added1-hydroxybenzotriazole (6.2 g, 0.04M) followed dropwise by a solution ofDCC (8.2 g, 0.04 mole) in CH₂ Cl₂. After 10 min. at 0° a solution ofphenethylamine (4.84 g, 0.04M) in CH₂ Cl₂ (15 ml) was added dropwise andthe stirred solution was then allowed to warm to room temperatureovernight. The reaction mixture was then filtered, concentrated in vacuoand dissolved in ethyl acetate (150 ml). The ethyl acetate solution waswashed in turn with water (40 ml), saturated aq. NaHCO₃ (50 ml×2),aqueous citric acid (50 ml) and saturated aq. NaHCO₃ (50 ml). Theresidue after evaporation of the solvent was recrystallised from ethylacetate/hexane to giveN-(tertiarybutoxycarbonyl)-L-leucyl-N-phenethylamide as a white powder(9.6 g); m.p. 86°-88°; ν_(max) (CHCl₃) 3415 and 1670 cm⁻¹ ; δ (CDCl₃)0.85 (6H,m,CH(CH₃)₂); 1.35 (9H,s,OC(CH₃)₃); 1.3-1.75 (3H,m,CH₂CH(CH₃)₂); 2.69 (2H,t,J=7.2 Hz, CH₂ C₆ H₅); 3.3-3.6 (2H,m,NCH₂); 4.05(1H,m,α-CH); 4.9 (1H,m,OCONH); 6.2 (1H,m,CONH); 7.2-7.4 (5H,m,C₆ H₅).

N-(tertiarybutoxycarbonyl)-L-leucine N-phenethylamide (6.17 g, mole) wasdissolved in a 1:1 TFA/CH₂ Cl₂ mixture (60 ml). After stirring for 6 hat 20° the reaction mixture was concentrated in vacuo and the residue inCH₂ Cl₂ (50 ml) washed with saturated aq. NaHCO₃ (100 ml). The aqueousextract was back extracted with CH₂ Cl₂ (50 ml×3) and the combinedorganic extracts concentrated to an oil in vacuo. The crude L-leucineN-phenethylamide so obtained was used as such in the next step.

To a solution of methyl 4-N-(benzyloxycarbonyl)amino-2-bromo-butanoate(330 mg, 1 mmole) in dry acetonitrile (10 ml) was added L-leucineN-phenethylamide (235 mg, 1 mM) and N-methyl morpholine (110 mg, 1 mM).The solution was heated at reflux overnight, sodium iodide (150 mg, 1mM) was added and the reaction was reheated to reflux for a further 7 h.The reaction mixture was then filtered and concentrated to an oil invacuo. Chromatography of the residue on silica in 1:1 EtOAc/Hexane gaveN-[3-N-(benzyloxycarbonyl)amino-1-(R,S)-methoxycarbonylpropyl]-L-leucineN-phenethylamide (310 mg). Rechromatography on silica then gave the Rdiastereoisomer as an oil.

To a solution of the foregoing R-isomer (110 mg) in methanol (4 ml) wasadded dilute NaOH (1N, 0.5 ml). After standing overnight at 20° thereaction mixture was acidified with acetic acid and concentrated to asolid in vacuo. Chromatography on reverse phase silica eluting with 1:1MeOH/H₂ O gave the title compound as a white powder (55 mg), m.p.130°-135°; (Found: C,65.62; H,7.59; N,8.85. C₂₈ H₃₅ N₃ O₅ +0.3H₂ Orequires C,65.75; H,7.55; N,8.85%); ν_(max) (Nujol) 1690, 1655 and 1630cm⁻¹ ; δ(d⁶ DMSO) 0.83 (6H,m,CH(CH₃)₂); 1.1-1.85 (6H,m,NCH₂ CH₂ CH₂CH(CH₃)₂ and NH); 2.69 (2H,t,J=7.2 Hz,CH₂ C₆ H₅); 3.0-3.6 (7H,NCH₂×2,α-CH×2, CO₂ H); 5.0 (2H,s,OCH₂ C₆ H₅); 7.1-7.5 (10H,m,C₆ H₅ ×2); 8.05(1H,m,CONH).

EXAMPLE 20 N-[5-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpentyl)-L-leucyl-O-methyl-L-tyrosine N-Methylamide

To a stirred solution of crude methyl6-N-(benzyloxycarbonyl)amino-2-oxo-hexanoate (7.03 g, 24 mM; Tet.Lett.,(1982), 23, 1875) and L-leucyl-O-methyl-L-tyrosine N-Methylamide (1.86g, 6 mM) in methanol (50 ml) was added acetic acid to bring the pH to6.5. Sodium cyanoborohydride (400 mg, 6.5 mM) was then added portionwisewhilst the pH of the solution was continually re-adjusted to 6.5 by theaddition of acetic acid. After 1.5 h at room temperature a furtherportion of sodium cyanoborohydride (400 mg) was added and the pH wasagain re-adjusted to 6.5 with acetic acid. After a further 1 h at roomtemperature, the reaction mixture was concentrated in vacuo and theresidue in CH₂ Cl₂ (50 ml) was washed in turn with water (30 ml), diluteHCl (1M, 30 ml) and saturated aq.NaHCO₃. The material isolated from theorganic layer was purified by column chromatography on silica in CH₂ Cl₂in an increasing ethyl acetate gradient to give the title compound as anoil (360 mg); (Found: [m+1]⁺ =xxx.xxxx. C₃₂ H₄₄ N₄ O₇ requires [m+1]⁺=xx.xxxx); δ (CDCl₃) 0.88 CH(CH₃)₂); 1.0-1.86 (10H,m,NHCH(CH₂)₃), CH₂CH(CH₃)₂ and NH); 2.74 (3H,d,J=5 Hz,NCH₃); 2.85-3.4 (6H,m,NHCH₂,CH₂ C₆H₄ and α-CH×2); 3.65 and 3.75 (each 3H, each s, 2×OCH₃); 4.64(1H,dd,J=13 Hz and 6 Hz, α-CH); 5.10 (2H,s,CH₂ C₆ H₅); 6.78 (2H,d,J=8.6Hz, Tyr H-3 and H-5); 7.10 (2H,d,J=8.6 Hz, Tyr H-2 and H-6); 7.35(5H,m,C₆ H₅) and 7.64 (1H,d,J=10 Hz,CONH).

EXAMPLE 21N-[5-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypentyl]-L-leucyl-O-methyl-L-tyrosineN-Methylamide

To a solution of the ester from Example 20 (140 mg, 0.23 mM) in methanol(10 ml) at 0° was added dilute NaOH (1N, 0.5 ml). After 48 h at 0°, afurther portion of NaOH (1N, 0.4 ml) was added and the solution stirredat 20° for a further 24 h. The reaction mixture was then acidified withacetic acid and concentrated in vacuo to give a semi-solid which waspurified by partition between ethyl acetate and water at 0°. Theresulting solid was filtered, washed with water and ethyl acetate andwas dried in vacuo to give the title compound (110 mg); 122°-128°;(Found: [m+1]⁺ =585.3290 C₃₁ H₄₄ N₄ O₇ requires [m+1]⁺ =585.3288)ν_(max) (Nujol) 3340, 1688 and 1640 cm⁻¹ ; δ(CD₃ OD) 0.88 (6H, m,CH(CH₃)₂); 1.0-1.86 (9H, m, NHCH₂ (CH₂)₃ and CH₂ CH(CH₃)₂); 2.74 (3H, s,NCH₃); 2.8-3.6 (6H, m, NHCH₂, CH₂ C₆ H₄ and α-CH×2); 3.77 (3H, s, OCH₃);4.60 (1H, m, α-CH); 5.10 (2H, s, CH₂ C₆ H₅); 6.78 (2H, d, J=8.6 Hz, TyrH-3 and H-5); 7.05 (1H, m, CONH); 7.10 (2H, d, J=8.6 Hz Tyr H-2 and H-6)and 7.35 (5H, m, C₆ H₅); m/e 585 (1%, [m+1]⁺), 567 (20% [m+1-H₂ O]⁺).

EXAMPLE 22N-[5-N-[N-Acetyl-L-prolyl]amino-1-(R)-carboxypentyl]-L-leucyl-O-methyl-L-tyrosineN-Methylamide

N-[5-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpentyl]-L-leucyl-O-methyl-L-tyrosineN-methylamide (400 mg, 0.66 mM) in methanol (20 ml) was treated withdilute HCl (1N, 1.2 ml) and PdCl₂ (50 mg). The reaction mixture wasstirred under hydrogen for 20 min. at room temperature and was thenfiltered. Concentration of the resulting solution in vacuo gaveN-[5-amino-1-(R)-methoxycarbonylpentyl]-L-leucyl-O-methyl-L-tyrosineN-methylamide hydrochloride as an oil. This was dissolved in CH₂ Cl₂ (20ml) and DMP (5 ml) and to the resulting solution was added N-methylmorpholine (300 mg) and N-acetyl-L-proline p-nitrophenyl ester (191 mg).After standing at 20° for 72 h, the reaction mixture was concentrated invacuo and the residue in ethyl acetate (20 ml) was washed with aq.citric acid solution. These aqueous washings were concentrated in vacuoand the resultant oil was purified by chromatography on reverse phasesilica eluting with a gradient of methanol in H₂ O to giveN-[5-N-(N-acetyl-L-prolyl)amino-1-(R)-methoxycarbonylpentyl]-L-leucyl-O-methyl-L-tyrosineN-methylamide (350 mg) δ(CDCl₃) 0.84 (6H, dd, J=14 Hz and 7 Hz,CH(CH₃)₂); 1.05-2.4 (13H, m, NHCH₂ (CH₂)₃, CH₂ CH(CH₃)₂ and CH₂ CH₂);2.08 (3H, s, COCH₃); 2.70 (3H, s, NCH₃); 2.76-3.82 (8H, m, NCH₂,NHCH₂ C₆H₄ and α-CH×2); 3.66 and 3.74 (each 3H, each s, 2×OCH₃); 4.32 (1H,m,α-CH); 4.56 (1H, dd, J=13 Hz and 6 Hz, α-CH); 6.80 (2H, d, J=8.6 Hz,tyr H-3 and H-5) and 7.12 (2H, d, J=8.6 Hz, Tyr H-2 and H-6).]

A portion of this material (130 mg) in methanol (5 ml) was treated at 0°with dilute NaOH (1N, 0.5 ml). After standing overnight at roomtemperature, a further portion of NaOH was added (1N, 0.2 ml) and thiswas then repeated 6 h later. After a further 18 h at 20° the reactionmixture was acidified with acetic acid and concentrated to an oil invacuo. Chromatography on reverse phase silica eluting with water in anincreasing methanol gradient gave the title compound (100 mg); m.p.97°-101°; (Found: [m+1]⁺ =590.3552C₃₀ H₄₇ N₅ O₇ requires [m+1]⁺=590.3554); ν_(max) (Nujol 3280 (br) and 1625 (br) cm⁻¹ ; δ(CD₃ CD) 0.94(6H, m, CH(CH₃)₂); 1.2-2.4 (13H, m, NHCH₂ (CH₂)₃, CH₂ CH(CH₃)₂ and CH₂CH₂); 2.12 (3H, s, COCH₃); 2.68 (3H, s, NCH₃); 2.75-4.1 (8H, m, NCH₂NHCH₂,CHHD 2C₆ H₄ and α-CH×2); 3.77 (3H, s, OCH₃); 4.33 and 4.58 (each1H, each m, 2×2CH); 6.85 (2H, d, J=8.6 Hz, Tyr H-3 and H-5); 7.16 (2H,d, J=8.6 Hz, Tyr H-2 and H-6) and 8.03 (1H, m, CONH); m/e 590 (2%,[m+1]⁺, 572 (10% [m+1-H₂ O]⁺).

EXAMPLE 23N-[2-(S)-N-(1-(R)-Carboxyethyl)amino-4,4-dimethylpentanoyl]-L-alanineN-Butylamide

N-[2-(S)-N(1-(R)-Methoxycarbonylethyl)amino-4,4-dimethylpentanoyl]-L-alanineN-butylamide (65 mg) in methanol (30 ml) was treated with IN-sodiumhydroxide (3 ml) at 20° for 6 h. Excess acetic acid was then added andthe solvent evaporated in vacuo. The residue was chromatographed onreverse phase silica (RF 18) in a gradient of 20%-80% methanol in water.Elution in 70% methanol in water afforded the title compound (30 mg) asa freeze-dried powder, m.p. 137°-138°; (Found: [m+1]⁺ =344.2548. C₁₇ H₃₄N₃ O₄ requires [m+1]⁺ =344.2549); δ(D₂ O) 0.9 (3H, t, J=6 Hz, CH₂ CH₃);0.94 (9H, s, C(CH₃)₃); 1.2-1.8 (6H, m, (CH₂)₂ and CH₂); 1.4 (3H, d, J=8Hz, CH₃ ); 1.52 (3H, d, J=7 Hz, CH₃); 3.18 (2H, t, J=6 Hz, NHCH₂); 3.66(1H, q, J=5 Hz, CHCO); 3.88 (1H, d, J=10 Hz, CHCH₂) and 4.38 (1H, q, J=5Hz, CHCH₃).

The starting material required in the preceeding preparation wassynthesised as described in the following paragraphs:

(a) Benzyl 2-Bromo-4,4-dimethylpentanoate

4,4-Dimethylpentanoic acid (40 g; Chem Lett, (1980), 571) was treated at20° for 16 h with thionyl chloride (40 g) and the mixture distilledunder reduced pressure to yield 4,4-dimethylpentanoyl chloride (38 g)b.p. 52°-58° at 10 mm Hg; δ (CDCl₃) 0.94 (9H, s, C(CH₃)₃ ; 1.66 (2H, t,J=9 Hz, CH₂) and 2.88 (2H, t, J=9 Hz, CH₂ CO).

A portion of this material (20 g) was treated at 110° with bromine (20g) for 4 h. Further bromine (5 g) was then added and the reactioncontinued for 1 h. Distillation under reduced pressure afforded2-bromo-4,4-dimethylpentanoyl chloride (26 g), b.p. 92°-96° at 10 mmHg;δ (CDCl₃) 1.0 (9H, s, C(CH₃)₃); 1.94 (1H, dd, J=15 and 5 Hz, CHCHBr);2.42 (1H, dd, J=15 and 8 Hz, CHCHBr) and 4.64 (1H, dd, J=8 and 5 Hz,CHBr).

The bromo-acid chloride (12 g) in CH₂ Cl₂ (100 ml) was treated withbenzyl alcohol (8.8 g) and N-methyl morpholine (4.06 g) at 0° for 16 h.The solution was then washed successively with dilute HCl andSat.aq.NaHCO₃ solution. The residue after evaporation of the solvent waspurified by chromatography on silica in 20% ether-hexane to give thedesired bromo ester (11.2 g) as an oil; (Found: C, 56.3; H,6.4; Br,26.8;C₁₄ H₁₉ Br,O requires C,56.2; H,6.4; Br,26.7%); ν_(max) 2940 and 1730cm⁻¹ δ(CDCl₃) 0.88 (9H, s, (CH₃)₃ C); 1.92 (1H, dd, J=15 and 4 Hz,CHCHBr); 2.38 (1H, dd, J=15 and 10 Hz, CHCHBr); 4.34 (1H, dd, J=10 and 4Hz CHBr); 5.2 (2H, s, OCH₂ -C₆ H₅) and 7.4 (5H, m, C₆ H₅).

(b) Benzyl2-(S)-N-(1-(R)-Methoxycarbonylethyl)amino-4,4-dimethylpentanoate

Benzyl-2-bromo-4,4-dimethylpentanoate (20 g) in dry dimethyl sulphoxide(250 ml) was treated with D-alanine methylester hydrochloride (9.33 g),N-methyl morpholine (6.78 g) and tetrabutyl ammonium iodide (24.7 g) at90° under an atmosphere of argon for 2 days. The reaction mixture wasallowed to cool to room temperature, poured into water (500 ml) and theproducts recovered by extraction into dichloromethane (3×250 ml). Thematerial isolated from the organic extracts was purified bychromatography on silica developed in a gradient of hexane-ether.Elution with 30% ether-hexane afforded benzyl 4,4-dimethylpent-2-enoate(14 g). Elution with 40% ether in hexane afforded the title compound(350 mg) as a gum; (Found: [m+1]⁺ =322.2022. C₁₈ H₂₇ N₁ O₄ requires[m+1]⁺ =322.2018); ν_(max) (film) 1735 cm⁻¹ ; δ(CDCl₃) 0.90 (9H, s,C(CH₃)₃ ); 1.28 (3H, d, J=7 Hz CHCH₃); 2.46 and 2.68 (2H, each dd, J=12and 5 Hz, CH₂ (CH₃)₃); 3.30 (1H, q, J=5 Hz CH-CH₃); 3.36 (1H, t, J=5 Hz,CH-CH₂); 3.66 (3H, s, OCH₃); 5.12 (2H, s, OCH₂) and 7.36 (5H, s, C₆ H₅).Elution with 45% ether in hexane afforded benzyl2-(R)-N-(1-(R)-methoxycarbonylethyl)-amino-4,4-dimethylpentanoate (340mg); (Found: [m+1]⁺ =322.2022. C₁₁ H₂₇ NO₄ requires 322.2018); ν_(max)(film) 3360 and 1735 cms⁻¹ ; δ(CDCl₃) 0.90 (9H, s, C(CH₃)₃); 1.28 (3H,d, J=6 Hz, CHCH₃ ); 1.44 and 1.72 (2H, each dd, J=5 and 12.5 Hz, CH₂);3.32 (1H, q, J=7 Hz, CHCH₃); 3.44 (1H, t, J=6 Hz, CHCH₂); 3.69 (3H, s,OCH₃), 5.24 (2H, s, OCH₂) and 7.36 (5H, m, C₆ H₅).

(c)N-[2-(S)-N(1-(R)-Methoxycarbonylethyl)amino-4,4-dimethylpentanoyl]-L-alanineN-Butylamide

The foregoing benzyl ester (450 mg) in methanol (50 ml) was treated withpalladium on charcoal (10% 400 mg) under 1 atmosphere of hydrogen withcontinuous stirring. When the uptake of hydrogen has ceased (15 min) thesolution was filtered and the filtrate concentrated in vacuo to afford2-(S)-N-(1-(R)-methoxycarbonylethyl)amino-4,4-dimethylpentanoic acid(210 mg); m.p. 120°-124° (from ether).

This material (200 mg) in CH₂ Cl₂ (50 ml) was treated with L-alanineN-butylamide hydrochloride (220 mg), N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (200 mg) and 1-hydroxybenzotriazole(120 mg) at 0° C. The pH of the reaction mixture was adjusted to 7 bythe addition of N-methyl morpholine. After 16 h at 20°, the solution waswashed in turn with saturated sodium hydrogen carbonate solution and 1Mcitric acid solution. The material isolated after evaporation of thedichloromethane was chromatographed on silica developed in a gradient of20% ethyl acetate in dichloromethane to 60% ethyl acetate indichloromethane to afford the title compound (110 mg) as a colourlessoil, (Found: [m+1]⁺ =358.2705. C₁₈ H₃₅ N₃ O₄ requires [m+1]⁺ =358.2706);(CDCl₃) 0.92 (3H, t, J=7.5 Hz, CH₂ CH₃); 1.0 (9H, s, C(CH₃)₃); 1.36 and1.40 (each 3H, each t, J=6 Hz, 2×CH₃); 1.2-1.9 (6H, m, 3×CH₂); 3.24 (2H,m, NHCH₂); 3.46 (1H, q, J=6 Hz, CH); 3.77 (3H, s, OCH₃), 4.46 (1H, t,J=6 Hz, CHCH₂); 4.5 (1H, q, J=6 Hz,CH), 7.15 (1H, m, NH) and 7.73 (1H,d, J=8 Hz, NH).

The L-alanine N-butylamide hydrochloride used in step (c) was preparedfrom N-tertiarybutoxycarbonyl-L-alanine N-butylamide by exposure to TFAin CH₂₃ Cl₂ followed by treatment with ethereal HCl. This in turn wasprepared from N-tertiarybutoxy-L-alanine and n-butylamine following theprocedure described in Example 2 forN-tertiarybutoxy-O-benzyl-L-tyrosine N-methylamide except thatbutylamine was used in place of methylamine hydrochloride.

EXAMPLE 24 N-(1-(R)-Carboxyethyl)-S-norleucyl-S-alanine. N-Butylamide

This was prepared from tertiarybutoxycarbonyl-L-norleucine, L-alanineN-butylamide and 2-bromopropionic acid methyl ester as described in thefollowing steps:

(a) Tertiarybutoxycarbonyl-L-norleucyl-L-alanine N-butylamide

Tertiarybutoxycarbonyl-L-norleucine (13.2 g) in CH₂ Cl₂ (200 ml) wastreated at 0° with L-alanine N-butylamide (5.25 g), DCC (7.77 g) and1-hydroxybenzotriazole (5 g). The pH of the reaction mixture wasadjusted to 7 with N-methyl morpholine and allowed to warm to roomtemperature overnight. The precipitated urea was filtered off and thefiltrate washed successively with saturated aqueous sodium hydrogencarbonate, water and 1M citric acid. The organic phase was dried oversodium sulphate and the solvent evaporated in vacuo. The residue waschromatographed on silica in a gradient of 30-70% ethyl acetate indichloromethane. Elution with 50% ethyl acetate in dichloromethaneafforded the title compound (7.6 g) which crystallised from ethylacetate as needles m.p. 108°-112°; (Found: C,60.8; H,9.8; N,11.8. C₁₈H₃₅ N₃ O₄ requires C,60.5; H,9.9; N,11.75%); ν_(max) (Nujol) 3280, 33401675 and 1640 cms⁻¹ δ (CDCl₃) 0.9 and 0.91 (each 3H, each t, each J=5Hz, 2×CH₃); 1.1-1.9 (10H, m, (CH₂)₃ and (CH₂)₂); 1.38 (3H, d, J=5 Hz,6H₂ CHCH₃); 1.44 (9H, s, C(CH₃)₃); 3.24 (2H, tt, J=5 Hz NHCH₂) and 4.1and 4.48 (each 1H, each m, 2×CH).

(b) L-Norleucine-L-alanine N-butylamide

Tertiarybutonycarbonyl-L-norleucine-L-alanine N-butylamide (5 g) indichloromethane (20 ml) was treated with trifluroacetic acid (20 ml) atroom temperature for 2 h. The solvents were evaporated in vacuo and theresidue in water was treated with excess sodium hydrogen carbonate andthe free amine recovered in dichloromethane. Evaporation of the CH₂ Cl₂and crystallisation of the residue from ether-hexane gave the titlecompound (3.1 g); m.p. 83°-84°; (Found: C,60.7; H,10.4; N,16.0. C₁₃ H₂₇N₃ O₂ requires C,60.6; H,10.6; N,16.3%); ν_(max) (Nujol): 3360, 3280,1635 and 1675 cm⁻¹ ; δ(CDCl₃) 0.94 (6H, t, J=5 Hz, 2×CH₂ CH₃); 1.40 (3H,d, J=6 Hz CH--CH₃); 1.4-1.9 (10H, m, (CH₂)₃ and (CH₂)₂); 3.26 (2H, dt,each J=5 Hz, NH--CH₂ --); 3.35 (1H, dd, J=4 and 8 Hz, CH--CH₂); 4.50(1H, dq, each J=6 Hz, CH--CH₃); 6.9 (1H, m, NH); 7.86 (1H, d, J=7Hz,NH).

(c) N-(1-(R)-Methoxycarbonylethyl)-S-norleucyl-S-alanine N-Butylamide

L-Norleucine-L-alanine N-butylamide (1 g) in acetonitrile (10 ml) wastreated with N-methyl morpholine (0.4 g) and methyl 2-bromopropionate(0.64 g) under reflux for 16 h. The solvent was removed in vacuo and theresidue in dichloromethane washed successively with 1M citric acid,water and saturated aqueous sodium hydrogen carbonate. The residue afterevaporation of the CH₂ Cl₂ was chromatographed on silica in gradient ofethyl acetate in CH₂ Cl₂. Elution with 60% ethyl acetate in CH₂ Cl₂afforded N-(1-(S)-methoxycarbonylethyl)-S-norleucyl-S-alanineN-butylamide (210 mg); (Found: [m+1]⁺ =344.2547. C₁₇ H₃₄ N₃ O₄ requires[m+1]⁺ =344.2582); ν_(max) (Nujol) 3320 and 1740 cms⁻¹ ; δ(CDCl₃) 0.95(6H, t, J=7 Hz, 2×CH₂ CH₃); 1.2-1.8 (10H, m, (CH₂)₂ and (CH₂ )₃); 2.98(1H, dd, J=4 and 5 Hz, CHCH₂); 3.24 (3H,m,NHCH₂ and CHCO); 3.7 (3H, s,OCH₃); 4.56 (1H, dq, J=5 Hz, CH) and 7.04 and 7.9 (each 1H, each m,2×NH).

Continued elution with 65% ethyl acetate in CH₂ Cl₂ gave the titlecompound (190 mg), m.p. 84°-88° (from ethyl acetate); (Found: C,59.2;H,9.5; N,12.2. C₁₇ H₃₃ N₃ O₄ requires C,59.6; H,9.4; N,12.3%); ν_(max)(Nujol) 3280 and 1740 cms⁻¹ ; δ(CDCl₃) 0.94 (6H, t, J=6 Hz, 2×CH₂ CH₂);1.38 and 1.42 (each 3H, each d, J=5 Hz, 2×CHCH₃); 1.3-1.9 (10H, m,(CH₂)₂); 3.06 (1H, dd, J=5 and 8 Hz, CHCH₂); 3.24 (2H, dt, J=5 and 6 Hz,NHCH₂); 3.46 (1H, q, J=6 Hz, CHCO); 3.72 (3H, s, OCH₃); 4.67 (1H, dq,J=5 and 7 Hz, CHCH₃); 6.84 (1H, m, NH) and 7.82 (1H, d, J=7 Hz, NH).

(d) N-(1-(R)-Carboxyethyl)-S-norleucyl-S-alanine. N-Butylamide

The foregoing methyl ester (150 mg) in CH₃ OH (50 ml) was treated with1M NaOH (1 ml) at room temperature for 72 h. Excess acetic acid wasadded and the solvents evaporated in vacuo. The residue waschromatographed on reverse phase silica (RP18) in a gradient of 0-60%methanol in water. Elution with 50% methanol in water afforded the titlecompound (110 mg) as needles from ether/hexane; m.p. 185°-190°; (Found:C,56.7; H,9.2; N,12.4. C₁₆ H₃₁ N₃ O₄.H₂ O requires C,56.8; H,9.5;N,12.4%); ν_(max) (Nujol) 3200 and 1650 cm⁻¹ ; δ(CD₃ OD) 0.92 and 0.94(each 3H, each t, each J=6 Hz, 2×CH₂ CH₃); 1.36 and 1.48 (each 3H, eachd, each J=6 Hz, 2×CHCH₃ ); 1.2-1.9 (10H, m, (CH₂)₂ and (CH₂)₃); 3.20(2H, t, J=6 Hz NH--CH₂); 3.56 (1H, q, J=6 Hz, CHCO₂ H); 3.88

EXAMPLE 25

(a)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypropyl]-L-leucyl-L-tyrosineN-Methylamide

To a stirred solution of the ester from example 11 (0.35 g, 0.63 mM) inmethanol (10 ml) was added dilute sodium hydroxide (1.3 ml, 1M). Afterstirring for 42 h at room temperature the reaction mixture wasneutralised with acetic acid, concentrated in vacuo and partitionedbetween ethyl acetate and water to give the title compound as a solid(121 mg); m.p. 179°-182°; (Found: [m+1]⁺ =557.2985. C₂₈ H₃₈ N₄ O₇requires 557.2975); δ(d⁶ DMSO) 0.8 (6H, m, CH(CH₃)₂); 1.18 (2H, m, CH₂CH(CH₃)₂); 1.46-1.9 (3H, m, NHCH₂ CH₂, CH₂ CH(CH₃)₂); 2.55 (3H, d, J=5Hz, CONHCH₃); 2.6-3.8 (6H, m, NHCH₂ CH_(2CHCO) ₂ H, α-CH, CH₂ Tyr); 4.38(1H, dd, J=7 and 15 Hz, α-CH); 5.02 (2H, s, CH₂ C₆ H₅); 6.63 (2H, d, J=8Hz, Tyr); 6.97 (2H, d, J=8 Hz, Tyr); 7.28 (1H, br, CONH); 7.36 (5H, s,C₆ H₅); 7.84 (1H, br, CONH); 8.14 (1H, d, J=12 Hz, CONH).

(b)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-L-tyrosineN-Methylamide

To a cold (0°) stirred solution ofN-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucine(422 mg, 1.01 mM) and N-methylmorpholine (204 mg) in dichloromethane (10ml) was added 1-hydroxybenzotriazole (154 mg, 1.01 mM) andN-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (194 mg,1.01 mM). After 15 minutes at 0° a solution of L-tyrosine N-methylamidehydrochloride (235 mg, 1.01 mM) in dichloromethane/dimethylformamide (11ml, 10:1) was added followed by N-methylmorpholine (102 mg, 1.01 mM).The reaction mixture was then allowed to warm and stir to roomtemperature overnight. The reaction mixture after washing with water, 3Ncitric acid, saturated aqueous sodium bicarbonate solution and water wasdried and concentrated in vacuo to a foam (0.35 g)δ(CDCl₃) 0.85 (6H, m,CH₂ (CH₃)₂); 1.1-2.0 (6H, m, NHCH₂ CH₂, CH₂ CH(CH₃)₂); 2.74 (3H, d, J=5Hz, CONHCH₃); 2.95-3.4 (6H, m, NHCH₂ CH₂ CH, α-CH, CH₂ C₆ H₄ ); 3.64(3H, s, OCH₃); 4.62 (1H, dd, J=7 and 15 Hz, α-CH); 5.10 (2H, s, CH₂ C₆H₅); 5.32 (1H, br, CONH); 6.78 (2H, d, J=8 Hz, C₆ H₄); 6.86 (1H, br,CONH); 7.02 (2H, d, J=8 Hz, C₆ H₄); 7.35 (5H, s, C₆ H₅); 7.65 (1H, d,J=10 Hz, CONH).

L-tyrosine N-methylamide hydrochloride used in this preparation wasprepared as follows:

(c) L tyrosine N-Methylamide hydrochloride

To a solution of O-benzyl-L-tyrosine N-methylamide HCl (1.13 g) inmethanol (50 ml) was added 10% Pd/C and the mixture was stirred in anatmosphere of hydrogen at room temperature and atmospheric pressure for4 h. The catalyst was then filtered and the solvent was removed in vacuoto yield L-tyrosine N-methylamide HCl as a foam (0.85 g).

(d)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucine

N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucinet-butyl ester (4.2 g) was treated with Trifluoroacetic acid (50 ml) andwater (5 ml) for one hour at room temperature. Volatiles were thenremoved in vacuo and the residue was co-evaporated with ethereal HCl.Recrystallisation of the residue from methanol/ether gave the titlecompound as its hydrochloride salt (2.2 g) m.p. 103°-109°; (Found: C,54.55; H, 7.37; N, 6.37. C₁₉ H₂₉ N₂ O₆ Cl requires C,54.74; H,7.01;N,6.72%); δ(d⁶ DMSO) 0.9 (6H, m, CH(CH₃)₂); 1.72 (3H, m,CH₂ CH(CH₃)₂);2.05 (2H, m, NHCH₂ CH₂); 3.12 (2H, m, NHCH₂); 3.74 (3H, s, OCH₃); 3.82(1H, m, α-CH); 4.02 (1H, m, α-CH); 5.02 (2H, s, CH.sub. 2 C₆ H₅); 7.35(5H, br s, C₆ H₅).

The foregoing t-Butyl ester was prepared as described below:

(e)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucinet-Butyl ester

To a solution of methyl 4-N-(benzyloxycarbonyl) amino-2-bromo-butanoate(64 g) and L-leucine t-butyl ester (34 g) in acetonitrile (300 ml) wasadded N-methylmorpholine (20 g) and the solution was heated under refluxfor 48 h. The reaction mixture was then cooled, concentrated in vacuo,taken up in water and extracted with ether. The combined etherealextracts were then dried and concentrated in vacuo to an oil. Columnchromatography of this oil on silica eluting with a gradient of ethylacetate in hexane gave the title compound as an oil (20 g); δ(CDCl₃)0.87 (6H, m, CH(CH₃)₂); 1.44 (9H, s, OC(CH₃)₃); 1.4-1.95 (5H, m, NHCH₂CH₂, CH₂ CH(CH₃)₂); 3.05-3.5 (4H, m, NHCH₂, α-CH×2); 3.70 (3H, s, OCH₃);5.10 (2H, s, CH₂ C₆ H₅ ); 5.73 (1H, m, CONH); 7.36 (5H, br s, C₆ H₅).

EXAMPLE 26

(a)N-(3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypropyl)-L-leucyl-O-propyl-L-tyrosineN-methyl amide

N-(3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl)-L-leucyl-O-propyl-L-tyrosineN-methyl amide (210 mg, 0.35 mM) in methanol (10 ml) was treated withaqueous sodium hydroxide (1.1 ml, 0.5M) at room temperature for 48 h.The reaction was then neutralised with acetic acid and concentrated invacuo to affordN-(3-N-Benzyloxycarbonyl)amino-1-(R)-carboxylpropyl)-L-leucyl-O-propyl-L-tyrosineN-methyl amide which crystallised from methanol/water as needles m.p.160°-167° C. (Found: C,63.2; H,7.8; N,9.7. C₃₁ H₄₄ N₄ O₇ +0.3H₂ Orequires: C,63.7; H,7.6; N,9.6%); δ(CDCl₃) 0.8 (9H, m, (CH₃)₂ CH,CH₃CH₂); 0.88-1.88 (9H, m, CH,CH₂,CH₂,CH₂ CH₂); 2.58 (3H, d, J=4 Hz CH₃NH); 2.2-3.66 (5H, m, CH₂,CH,CH₂); 3.84, (2H, t, J=6 Hz, CH₂ --O); 4.4(1H, m, NHCHCO); 5.02 (2H, s, O--CH₂ C₆ H₅); 5.6 (1H, m, NH); 6.76, and7.08 (4H, each d, each J=7 Hz, C₆ H₄); 7.24 (1H, m, NH); 7.32 (5H, m, C₆H₅); 7.8 (1H, m, NH); 8.14 (1H, m, NH).

The preceeding methyl ester was prepared as follows:

(b)N-(3-N-(Benzyloxycarbonyl)amino-1-(R)methoxycarbonypropyl)-L-leucyl-O-propyl-L-tyrosineN-methyl amide

N-Tertiary butyloxycarbonyl-O-propyl-L-tyrosine N-methyl amide (370 mg,1.1 mM) in dichloromethane (8 ml) was treated with trifluoroacetic acid(2 ml) at 20° C. for 2 h. The solvent was removed in vacuo and theresidue redissolved in ether saturated with hydrogen chloride and thisprocedure repeated twice more to afford O-propyl-L-tyrosine-N-methylamide hydrochloride (1.1 mM) which was used directly in the next step.N-(3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl)-L-leucinehydrochloride (prepared as described in Example 1) (416 mg, 1 mM) indichloromethane (10 ml) and dimethyl formamide (2 ml) was treated with1-hydroxybenzotriazole (159 mg, 1.04 mM), O-propyl-L-tyrosine-N-methylamide hydrochloride (1.1 mM), N-methyl morpholine (315 mg, 3.3 mM) andN-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (200 mg,1.04 mM) at 0° C. The reaction mixture was stirred continuously as itwas allowed to warm to 20° C. over 16 h. The reaction was diluted withdichloromethane (30 ml) and washed successively with water, aqueoussaturated sodium hydrogen carbonate and aqueous citric acid (1M) driedover anhydrous sodium sulphate and concentrated in vacuo to affordN-(3-N-Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl)-L-leucyl-O-propyl-L-tyrosine-N-methylamide (0.22 g, 0.36 mM) which crystallised from ethyl acetate/hexane asneedles m.p. 135°-140° C. (Found: C,62.8; H,7.8; N,9.2. C₃₂ H₄₆ N₄ O₇+0.5 H₂ O requires: C,63.2; H,7.8; N,9.2%); δ(CDCl₃) 0.86 (6H,d,d,J=4 HzJ=4 Hz (CH₃)₂ C); 1.02 (3H,t,J=7 Hz CH₃ CH₂); 1.1-1.98(9H,m,CH,CH₂,CH₂,CH₂ CH₂); 2.74 (3H,d,J=4 Hz CH₃ NH); 2.86-3.48,(5H,m,CH₂ CH₂,CH); 3.64 (3H,s,OCH₃); 3.86 (2H,t,J=6 Hz,CH₂ O); 4.58(1H,m,NHCHCO); 5.06 (2H,s,CH₂ C₆ H₅); 5.14 (1H,m,NH) 6.54 (1H,m,NH);6.76, and 7.06 (4 H,each d, each J=8 Hz,C₆ H₄); 7.32 (5H,m,C₆ H₅); 7.42(1H,m,NH).

The intermediate used in the preceeding section was prepared as follows:

(c) N-Tertiarybutyloxycarbonyl-O-propyl-L-tyrosine N-methyl amide

Tertiarybutyloxycarbonyl-L-tyrosine (14.1 g, 50 mM) in dimethylformamide (200 ml) was cooled to 10° C. and treated with sodium hydride(80%, 3.3 g, 110 mM) with continuous stirring for 1 h. Propyl bromide(6.15 g, 50 mM) was added and the reaction allowed to warm to 20° C.over 16 h. Water (50 ml) was added and the reaction concentrated invacuo to 100 ml, water 300 ml was added and the solution washed twicewith ethyl acetate (300 ml). The reaction was then acidified to pH1 withhydrochloric acid (6N) and extracted twice with ethyl acetate. Theorganic extract was dried over anhydrous sodium sulphate andconcentrated in vacuo to a gum. Column chromatography on silica in ethylacetate afforded N-tertiarybutyl oxycarbonyl-O-propyl-L-tyrosine (15.1 g46.7 mM) as a foam which was used directly in the next step. To (13 g,40 mM) of this foam in dichloromethane (250 ml) was added1-hydroxybenzotriazole (6.8 g, 45 mM); methylamine hydrochloride (2.5 g,80 mM); N-methyl morpholine (8 g, 80 mM) and dicyclohexylcarbodiimide(9.1 g, 45 mM) at 0° C. The reaction was stirred continuously whilstallowed to warm to 20° C. over 6 h. The reaction was filtered and thefiltrate washed with water, aqueous saturated sodium hydrogen carbonateand aqueous citric acid (1M), dried over anhydrous sodium sulphate andconcentrated in vacuo to affordN-tertiarybutyloxycarbonyl-O-propyl-L-tyrosine -N-methyl-amide (8.5 g,25 mM) which crystallised from ethyl acetate as needles m.p. 134°-135°(Found: C,64.5; H,8.7; N,8.4. C₁₈ H₂₈ N₂ O₄ requires: C,64.3; H,8.4;N,8.3%; δ(CDCl₃) 0.96 (3H,t,J=8 Hz,CH₃ CH₂); 1.36 (9H,s,(CH₃)₃ C); 1.74(2H,t,q,J=7 Hz,J=7 Hz,CH₂); 2.68 (3H,d,J=4 Hz,CH₃ NH); 2.92 (2H,m,CH₂ C₆H₄); 3.82 (2H,t,J=7 Hz,CH₂ -O); 4.16 (1H,m,NHCHCO); 4.98 (1H,m,NH); 5.68(1H,m,NH); 6.74 and 7.0 (4H, each 2H, each d, each J=8 Hz,C₆ H₄).

EXAMPLE 27

(a)N-[3-N-(Benzyloxycarboxyl)amino-1-(R)-carbonypropyl]-L-leucyl-O-isopropyl-L-tyrosineN-methylamide

N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl)-L-leucyl-O-isopropyl-L-tyrosineN-methylamide (0.4 g 0.65 mM) in methanol (15 ml) was treated withaqueous sodium hydroxide (2 ml, 0.5M, 0.75 mM) at 20° C. for 24 h. Thesolution was then adjusted to pH7 with acetic acid and concentrated tolow volume. The white solid which crystallised was recrystallised frommethanol/water 1:1 to affordN-[3-N-Benzyloxycarbonyl)amino-1-(R)-carboxypropyl]-L-leucyl-O-isopropyl-L-tyrosine-N-methylamide (100 mg, 0.17 mM) m.p. 148°-160° C. (Found: C,63.3; H,7.6; N,9.6.C₃₁ H₄₄ N₄ O requires: C,63.7; N,7.8; N,9.3%) δ(CDCl₃) 0.8 (6H,m, (CH₃)₂C); 0.98-1.84 (5H,m,CH₂,CH₂ CH); 1.22 (6H,d,J=6 Hz, (CH₃)₂ CHO); 2.56(3H,d,J=4 Hz CH₃ NH); 2.64-3.66 (7H,m,CH₂,CH₂,CH₂ ,CH); 4.4(1H,m,NHCHCO); 4.52 (1H,m,NHCHCO); 5.0 (2H,s,OCH₂ C₆ H₅); 6.72, and 7.06(each 2H,each d,each J=8 Hz C₆ H₄); 7.32 (5H,m,C₆ H₅); 7.88 (1H,m,NH);8.22 (1H,m,NH).

The preceeding methyl ester was prepared as follows:

(b)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-isopropyl-L-tyrosineN-methylamide

N-Tertiary butyloxycarbonyl-O-isopropyl-L-tyrosine-N-methylamide (370mg, 1.1 mM); in dichloromethane (10 ml) was treated with trifluoroaceticacid (10 ml) at 20° C. for 1 h. The resulting trifluoroacetate wasconverted to the hydrochloride salt by concentrating to a gum in vacuoand redissolving the residue in ether saturated with hydrogen chloride.This procedure was repeated three times. To the product indichloromethane (10 ml) was addedN-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucinehydrochloride (416 mg, 1 mM) (prepared as described in Example 1)1-hydroxybenzotriazole (159 mg, 1.04 mM), andN-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (200 mg,1.04 mM) at 0° C. The stirred reaction mixture was adjusted to pH7 withN-methylmorpholine and allowed to warm to room temperature over 16 h.The reaction was diluted with dichloromethane and washed with water,aqueous saturated sodium hydrogen carbonate, and aqueous citric acid(1M), dried over sodium sulphate and concentrated in vacuo to a gum.(Found: [M+H]⁺ = 599.3446. C₃₂ H₄₆ N₄ O₇ requires: [M+H]⁺ =599.3445);δ(CDCl₃) 0.84 (6H,m,(CH₃)₂ CH) 1.3 (6H,d,J=7 Hz, (CH₃)₂ CHO) 1.1-1.96(5H,m,2×CH₂,CH) 2.72 (3H,d,J=4 Hz,CH₃ NH); 3.04-3.36 (6H,m,2×CH₂,2×CH);3.64 (3H,s,CH₃ O); 4.46 (1H,m,NHCHCO); 4.56 (1H,m,NHCHCO); 5.06(2H,s,O--(CH₂ C₅ H₆); 5.32 (1H,m,NH); 6.76, and 7.04 (each 2H,eachd,each J=8 Hz,C₆ H₄); 6.98 (1H,m,NH); 7.3 (5H,m,C₆ H₅); 7.52 (1H,m,NH).

The intermediate used in the preceeding section was prepared as follows:

(c) N-Tertiarybutyloxycarbonyl-O-isopropyl-L-tyrosine N-methylamide

N-Tertiarybutyloxycarbonyl-L-tyrosine (14.1 g 50 mM) in drydimethylformamide (200 ml) was treated with sodium hydride (3.45 g, 115mM) with vigorous stirring under an atmosphere of argon at 10° C. for 1h. 2-Bromopropane (6.15 g, 50 mM) was added and the stirred solutionallowed to warm to 20° C. over 16 h. Water (200 ml) was added and thesolution extracted with ethyl acetate (2×200 ml). The reaction wasadjusted to pH1 with hydrochloric acid (6M) and extracted withdichloromethane 2×300 ml. The combined extracts were columned on silicain ethyl acetate to affordN-tertiarybutyloxycarbonyl-O-isopropyl-L-tyrosine (13.6 g, 42 mM) as afoam. The foam was dissolved in dichloromethane (200 ml) and treatedwith methylamine hydrochloride (2.5 g, 80 mM), 1-hydroxybenzotriazole(6.75 g, 44 mM), dicyclohexylcarbodiimide (9.1 g, 44 mM) and N-methylmorpholine (2.5 g, 80 mM) at 0° C. with continuous stirring. Thesolution was allowed to warm to room temperature over 16 h and filtered.The filtrate was washed with water, aqueous saturated sodium hydrogencarbonate solution and aqueous citric acid (1M), dried over sodiumsulphate and concentrated in vacuo to affordN-tertiarybutyloxycarbonyl-O-isopropyl-L-tyrosine-N-methyl amide (6.1 g,18 mM) which crystallised from ethyl acetate/hexane as needles m.p.110°-114° C. (Found: C,63.5; H,8.4; N,8.7. C₁₈ H₂₈ N₂ O₄ 0.25 H₂ Orequires: C,63.4; H,8.4; N,8.2%) δ(CDCl₃) 1.32 (6H,d,J=7 Hz, (CH₃)₂ CH);1.4 (9H,s,(CH₃)₃ C); 2.72 (3H,d,J=4 Hz NHCH₃); 2.98, (2H,d,J=7 Hz CH₂ C₆H₅); 4.26 (1H,m,NHCHCO); 4.5 (1H, heptet, J=5 Hz, CH(CH₃)₂); 5.08(1H,m,NH); 5.92 (1H,m,NH); 6.78, and 7.04 (each 2H,each d,each J=8 Hz C₆H₄).

EXAMPLE 28

(a) N-[3-N-(Benzyloxycarboxyl)(amino-1-(R)-carboxypropyl)-L-leucyl-Otertiary butyl-L-tyrosine. N-methyl amide

N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-tertiarybutyl-L-tyrosine N-methyl amide (0.26 g, 0.42 mM) in methanol (10 ml)was treated with aqueous sodium hydroxide (1.3 ml, 0.5M) at 20° C. for24 h. The reaction was then adjusted to pH 7 with hydrochloric acid (6N)and concentrated in vacuo to low volume. The white solid whichcrystallized out was washed with ether and water and dried in vacuo toafford N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypropyl-L-leucyl-O-tertiary butyl-L-tyrosine-N-methyl amide m.p. 160° C.dec. (Found: C,62.51; H,7.67; N,9.24; C₃₂ H₄₆ N₄ O₇.H₂ O requires:C,62.32; H,7.84; N,9.08%). δ(CDCl₃) 0.78 (6H,m (CH₃)₂ C); 1.08-1.86(5H,m,CH₂,CH₂ CH); 1.24 (9H,s,(CH₃)₃ C); 2.56 (3H,d,J=4 Hz,CH₃ NH);2.44-3.2 (6H,m,2×CH₂,2×CH); 4.4 (1H,m,NHCHCO); 5.0 (2H,s,CH₂ C₅ H₆);6.82 and 7.08 (each 2H,each d,each J=8 Hz,C₆ H₄); 7.22 (1H,m,NH); 7.3(5H,m,C₆ H₅); 7.84 (1H,m,NH); and 8.2 (1H,m,NH).

The preceeding methyl ester was prepared as follows:

(b) N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxy carbonylpropyl]-L-leucyl-O-tertiary butyl-L-tyrosine N-methyl amide

N-Benzyloxycarbonyl-O-tertiarybutyl-L-tyrosine N-methylamide (2.4 g 6.2mM) in ethanol (10 ml) was heated under reflux with cyclo hexene (4 ml)acetic acid (0.38 g 6.2 mM) and palladium on carbon (10%, 1 g) under anatmosphere of argon for 20 m. The solvents were then removed in vacuo toafford O-tertiarybutyl-L-tyrosine N-methylamide acetate m.p. 121°-123°C. This was dissolved in dichloromethane (50 ml) and shaken with aqueoussaturated sodium hydrogen carbonate (50 ml). The organic layer was driedover anhydrous sodium sulphate and concentrated in vacuo to affordO-tertiary butyl-L-tyrosine N-methyl amide as a crude gum which was useddirectly in the next step. N-[3-N-Benzyloxycarbonyl]amino-1-(R)-methoxycarbonylpropyl]-L-leucine hydrochloride (418mg, 1 mM) in dichloromethane (10 ml) was treated with1-hydroxybenzotriazole (159 mg, 1.0 mM) N-methylmorpholine (210 mg, 2mM), O-tertiarybutyl-L-tyrosine-N-methyl amide (275 mg, 1.1 mM) andN-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride (200 mg,1.04 mM) at 0° C. with continuous stirring. The reaction mixture wasadjusted to pH7 with N-methylmorpholine and was allowed to warm to 20°C. over 16 h. The reaction was diluted with dichloromethane and washedsuccessively with water, aqueous saturated sodium hydrogen carbonate andaqueous 3N citric acid, dried over sodium sulphate and concentrated invacuo to afford N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonypropyl]-L-leucine-O-tertiary butyl-L-tyrosine-N-methyl amide (350mg, 0.6 mM) which crystallised from ethyl acetate/hexane as needles m.p.66°-68° C. (Found: C,64.7; H,8.1; N,9.2. C₃₃ H₄₈ N₄ O₇ requires: C,4.7;H,7.9; N,9.1%); δ(CDCl₃) 0.88 (6H,d,J=7 Hz, (CH₃)₂ C); 1.32 (9H,s,(CH₃)₃ C); 1.16-2.0 (5H,m,CH₂,CH₂ CH); 2.72 (3H,d,J=5 Hz, CH₃ NH); 3.02to 3.4 (6H,m,2×CH,2×CH₂); 3.66 (3H,s,CH₃ O); 4.58 (1H,m,NHCHCO); 5.08(2H,s,CH₂ C₅ H₆); 5.2 (1H,m,NH); 6.54 (1H,m,NH); 6.88 and 7.06 (each2H,each d,each J=8 Hz,C₆ H₄); 7.32 (5H,m,C₆ H₅); 7.52 (1H,m,NH).

The foregoing intermediate was prepared as follows:

(c) N-Benzyloxycarbonyl-O-tertiarybuty-L-tyrosine N-methyl amide

Benzyloxycarbonyl-O-tertiarybutyl-L-tyrosine dicyclohexyclamine salt (5g, 0.9 mM) was dissolved in aqueous citric acid (25 ml, 1M) andextracted with ethyl acetate (3×50 ml). The organic layer was dried oversodium sulphate and concentrated in vacuo to afford the free acid (4.1g) as a gum. This was dissolved in dichloromethane (100 ml) and treatedwith hydroxy benzotriazole (1.5 g, 10 mM), methylamine hydrochloride(0.56 g, 18 mM) and dicyclohexyl carbodiimide (2.1 g 10 mM) at 0° C.with continuous stirring. The reaction was allowed to warm to 20° C.over 16 h and then filtered. The filtrate was diluted withdichloromethane and washed with water. aqueous saturated sodium hydrogencarbonate and aqueous citric acid (1M), dried over sodium sulphate andconcentrated in vacuo to afford Benzyloxycarbonyl-O-tertiarybutyl-L-tyrosine N-methylamide (2.5 g) which crystallised from ethylacetate/hexane as needles m.p. 124°-125° C. (Found: C,68.7; H,7.9;N,7.4. C₂₂ H₂₈ N₂ O₄ requires: C, 68.7, H,7.3; N,7.3%); δ(CDCl₃) 1.34(9H,s,(CH₃)₃ C); 2.68 (3H,d,J=4 Hz NHCH₃); 2.94 and 3.1 (2H,each dd,eachJ=15 Hz,J=6 Hz,CH₂ C₆ H₄); 4.3 (1H,q,J=5 Hz NHCHCO); 5.08 (2H,s,CH₂ C₆H₅); 5.34 (1H,m,NH); 5.58 each (1H,m,NH); 6.88, and 7.06 (each 2H,eachd, each J=8 Hz,C₆ H₄); 7.3 (5H,m,C₆ H₅).

EXAMPLE 29

(a)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypropyl]-L-leucyl-O-pentyl-L-tyrosineN-Methylamide

To a stirred solution of 1 (280 mg, 0.47 mM) in methanol (10 ml) wasadded dilute sodium hydroxide (0.7 ml, 1M). After stirring for 48 h atroom temperature the reaction was neutralised with acetic acid and wasconcentrated in vacuo to a solid. Recrystallisation from methanol/watergave the title compound as a solid (107 mg); m.p. 165°-170°; δ(d⁶ DMSO)0.8 (6H, dd, J=5 Hz, J=CH₂ CH(CH₃)₂); 0.87 (3H, t, J=7 Hz, CH₂ CH₃);1.1-1.25 (2H, m, CH₂ CH(CH₃)₂); 1.26-1.85 (9H, m, OCH₂ (CH₂)₃ (CH₃),NHCH_(2CH) ₂ CH, CH₂ CH(CH₃)); 2.56 (3H, d, J=8 Hz, CONHCH₃); 2.6-3.65(6H, m, NHCH₂ CH₂ CH CO₂ H, α-CH, CH₂ Tyr); 3.87 (2H, t, J=7 Hz, OCH₂CH₂); 4.42 (1H, dd, J=7 and 15 Hz, α-CH); 5.02 (2H, s, CH₂ C₆ H₅); 6.77(2H, d, J=8 Hz, Tyr); 7.10 (2H, d, J=8 Hz, Tyr); 7.26 (1H, br, CONH);7.35 (5H, s, C₆ H₅); 7.85 (1H, br, CONH); 8.14 (1H, d, J=8 Hz, CONH).

The preceeding ester was synthesised as described below:

(a) N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-pentyl-L-tyrosine N-Methylamide

To a solution ofN-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucinehydrochloride (418 mg, 1 mM) in dichloromethane (10 ml), stirred andcooled to 0° was added N-methylmorpholine (101 mg),1-hydroxybenzotriazole (153 mg, 1 mM) and dicyclohexylcarbodiimide (206mg, 1 mM). After 15 minutes at 0° a solution of O-pentyl-L-tyrosineN-methylamide (265 mg) (which had been prepared from theN-benzyloxycarbonyl precursor in the usual manner) was added dissolvedin dichloromethane (10 ml). The reaction after being allowed to warm andstir to room temperature overnight was filtered, washed with water, 3Ncitric acid, saturated aqueous sodium bicarbonate and water. The washedorganic extract was dried and concentrated to an oil in vacuo.Chromatography on silica eluting with ethyl acetate gaveN-[3-N-(benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-pentyl-L-tyrosine N-Methylamide as a solid (359 mg);m.p. 81°-85°; (Found: C, 64.78; H, 8.05; N,8.82. C₃₄ H₅₂ N₄ O₇ requiresC, 64.94; H, 8.34; N, 8.91%); δ(CDCl₃) 0.83 (3H, d, J=5 Hz, CH₂ CH(CH₃)0.87 (3H, d, J=5 Hz, CH₂ CH(CH₃); 0.92 (3H, d, J=7 Hz, CH₂ CH₃); 1.0-2.0(12H, NHCH₂ CH₂, NH, CH₂ CH(CH₃)₂, OCH₂ (CH₂)₃ CH₃); 2.75 (3H, d, J=5Hz, CONHCH₃); 2.9-3.6 (6H, m, NHCH₂ CH₂ CHCO₂ H, α-CH, CH₂ Tyr); 3.66(3H, s, OCH₃); 3.9(2H, t J=7 Hz, OCH₂ CH₂); 4.58 (1H, dd, J=7 and 15 Hz,PG,106 CH); 5.11 (3H, br, CH₂ C₆ H₅, CONH); 6.58 (1H, br, CONH); 6.81(2H, d, J=8 Hz, C₆ H₄); 7.10 (2H, d, J=8 Hz, C₆ H₄); 7.36 (5H, s, C₆H₅); 7.47 (1H, d, J=8 Hz, CONH).

N-Benzyloxycarbonyl-O-pentyl-L-tyrosine N-methylamide used in thispreparation was synthesized as follows:

(c) N-(Benzyloxycarbonyl)-O-pentyl-L-tyrosine N-Methylamide

To a cold (0°) stirred solution ofN-benzyloxycarbonyl-O-pentyl-L-Tyrosine (8.3 g, 22 mM) in THF (120 ml)was added 1-hydroxybenzotriazole (3.6 g, 24 mM) anddicyclohexylcarbodiimide (4.9 g, 24 mM). After stirring for 25 minutesat 0° a solution of methylamine in THF (7.2 ml, 5M) was added and thereaction was stirred and warmed to room temperature overnight. Thereaction mixture was then filtered, evaporated in vacuo and dissolved indichloromethane (150 ml). The dichloromethane extract after washing withwater, 3N citric acid, saturated sodium bicarbonate solution and waterwas dried, filtered and concentrated in vacuo to a solid.Recrystallisation from ethyl acetate gave the required N-Methylamide asneedles (6.43 g); m.p. 132°-135°; (Found: C, 69.30; H, 7.63; N, 7.43.C₂₃ H₃₀ N₂ O₄ requires C, 69.32; H, 7.59; N, 7.03%); δ(CDCl₃) 0.92 (3H,t, J=7 Hz, CH₂ CH₃); 1.25-2.0 (6H, m, OCH₂ (CH₂)₃ CH₃); 2.71 (3H, t, J=5Hz, CONHCH₃); 3.0 (2H, m, CH₂ Tyr); 3.92 (3H, t, J=7 Hz, OCH₂ CH₃); 4.29(1H, dd, J=7 and 15 Hz, α-CH); 5.09 (2H, s, CH₂ C₆ H₅); 5.37 (1H, br,CONH); 5.69 (1H, br, CONH); 6.82 (2H, d, J=8 Hz, Tyr); 7.08 (2H, d, J=8Hz, Tyr); 7.33 (5H, m, C₆ H₅).

The preceeding acid was synthesized as described below.

(d) N-(Benzyloxycarbonyl)-O-pentyl-L-tyrosine

To a cold (10°) stirred solution of N-benzyloxycarbonyl-L-tyrosine(12.61 g, 40 mM) in dry DMF (150 ml) under argon was added sodiumhydride (2.76 g, 80% dispersion in oil). After stirring for one hour at10° n-bromopentane (6.0 g, 40 mM) was added and the reaction mixture wasallowed to stir and warm to room temperature overnight. Water (800 ml)and ethyl acetate (800 ml) were then added and the separated organicphase was acidified and extracted with ethyl acetate (200 ml×3). Thisorganic extract was dried, filtered and concentrated to an oil in vacuo.Chromatography on silica eluting with ethyl acetate gaveN-benzyloxycarbonyl-O-pentyl-L-Tyrosine which was recrystallised fromether/hexane to give a solid (8.5 g), m.p. 77°-81°; (Found: C, 68.58; H,7.19; N, 3.77. C₂₂ H₂₇ NO₅ requires C, 68.55; H, 7.06; N, 3.63%); δ(CDCl₃) 0.92 (3H, t, J =7 Hz, CH₂ CH₃); 1.29-1.52 (4H, m, CH₂ alkyl);1.66-1.86 (2H, m, CH₂ alkyl); 3.0-3.12 (2H, m, CH₂ Tyr); 3.93 (2H, t,J=7 Hz, OCH₂ CH₃); 4.67 (1H, m, CH); 5.12 (2H, s, CH₂ C₆ H₅); 5.2 (1H,d, J-8 Hz, CONH); 6.82 (2H, d, J=8 Hz, C₆ H₄); 7.07 (2H, d, J=8 Hz, C₆H₄); 7.35 (5H, s, C₆ H₅).

EXAMPLE 30

(a)N[3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypropyl]-L-leucyl-O-3(methyl)butyl-L-tyrosineN-methyl amide

N-[3-N-(Benzyloxycarbonyl)amino-1-(R)methoxycarbonylpropyl]-L-leucyl-O-3(methyl)butyl-L-tyrosineN-methyl amide (270 mg 0.43 mM) in methanol (10 ml) was treated withaqueous sodium hydroxide (1.3 ml, 0.5M) at 20° C. for 48 h. The reactionwas adjusted to pH4 with acetic acid and the solvent removed in vacuo toaffordN-[3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypropyl]-L-leucyl-O-3(methyl)butyl-L-tyrosineN-methylamide (121 mg) which crystallised from methanol/water as needlesm.p. 155°-160° C. (Found: C,63.7; H,7.7; N,9.2. C₃₃ H₄₈ N₄ O₇ requires:C,64.7; H,7.9; N,9.1%); δ ((CD₃)₂ SO) 0.8 and 0.92 (12H,each m, 2×(CH₃)₂C); 1.0-1.9 (8H,m,3×CH₂,2×CH) 2.56 (3H,d,J=4 Hz CH₃ NH) 2.76-3.8(6H,m,2×,CH₂,2×CH); 3.92 (2H,m,CH₂ O); 4.52 (1H,m,NHCHCO); 5.0 (2H,s,CH₂C₆ H₅); 6.76 and 7.1 (each 2H,each d,each J=8 Hz,C₆ H₄); 7.34 (5H,m,C₆H₅); 7.9 (1H,m,NH); 8.1 (1H,m,NH).

The preceeding methyl ester was prepared as described below:

(b) N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-3(methyl)butyl-L-tyrosine N-methyl amide

Tertiarybutyloxycarbonyl-O-3-methylbutyl-L-tyrosine-N-methylamide (401mg, 1.1 mM) in dichloromethane (10 ml) was treated with trifluoroaceticacid (10 ml) at 20° C. for 1.5 h. The solution was concentrated in vacuoto a gum and dissolved in ether saturated with hydrogen chloride, andthis procedure repeated twice to affordO-3(methyl)butyl-L-tyrosine-N-methylamide hydrochloride which was useddirectly in the next step.N(3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonyl propyl)-L-leucineHydrochloride (418 mg 1 mM) in dichloromethane (20 ml) and dimethylformamide (2 ml) was treated with N-methyl morpholine (420 mg, 4 mM),1-hydroxybenzo-triazole (159 mg 1.04 mM), O-3-Methylbutyl-L-tyrosineN-methyl amide hydrochloride (1.1 mM) andN-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (200 mg,1.04 mM) at 0° C. with continuous stirring. The reaction was allowed towarm to 20° C. over 16 h. The reaction was diluted with dichloromethane(30 ml) and washed successively with water, aqueous saturated sodiumhydrogen carbonate and aqueous citric acid (1M), dried over sodiumsulphate and concentrated in vacuo to affordN-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonypropyl)-L-leucyl-O-3(methyl)butyl-L-tyrosine N-methylamide (360mg, 0.57 mM) which crystallised from ethyl acetate/hexane as needlesm.p. 78°-81° C. (Found: C,65.8; H,8.0; N,8.9 C₃₄ H₅₀ N₄ O₇ requires:C,64.8; H,8.3; N,8.9); δ (CDCl₃) 0.86 (6H,dd,J=6 Hz J=1 Hz (CH₃)₂ C)0.94 (6H,d,J=6 Hz, (CH₃)₂ C); 1.02-2.0 (8H,m,3×(CH₂),2×CH); 2.74(3H,d,J=4 Hz,CH₃ NH); 2.92-3.42 (6H,m,2×CH₂,2×CH); 3.66 (3H,s,CH₃ O);3.92 (2H,t,J=6 Hz,CH₂ O); 4.58 (1H,m,NHCHCO); 5.1 (2H,s,CH₂ C₆ H₅); 5.16(1H,m,NH); 6.52 (1H,m,NH); 6.78 and 7.06 (each 2H, each d, each J=8Hz,C₆ H₄); 7.3 (5H,m,C₆ H₅); 7.74 (1H,m,NH).

The intermediate used in the preceeding section was prepared asdescribed below:

(c) Tertairybutyl-oxycarbonyl-O-3(methyl)butyl-L-tyrosine-N-methylamide

Tertiarybutyloxycarbonyl-L-tyrosine (14.1 g, 50 mM) in dry dimethylformamide (200 ml) was treated with sodium hydride (3.45 g, 115 mM)under an atmosphere of argon at 10° C. for 1 h. 3(Methyl)butyl bromide(8.3 g, 55 mM) was added and the reaction allowed to warm to 20° C. over20 h. Water (200 ml) was added and the solution washed with ethylacetate (2×300 ml) and adjusted to pH1 with 6N HCl. The solution wasextracted with ethyl acetate and the extract dried over sodium sulphateand concentrated in vacuo to afford tertiary butyloxycarbonyl-O-3(methyl)butyl-L-tyrosine (15 g, 35 mM) as a gum. The gumwas dissolved in dichloromethane (200 ml) and treated with1-hydroxybenzotriazole (5.8 g, 38 mM) methylamine hydrochloride (2.2 g,70 mM), dicyclohexyl carbodiimide (7.8 g, 38 mM), adjusted to pH7 withN-methyl morpholine and stirred continuously as the reaction was allowedto warm from 0° C. to 20° C. over 16 h. The reaction was filtered andwashed with water, aqueous saturated sodium hydrogen carbonate andaqueous saturated citric acid, dried over sodium sulphate andconcentrated in vacuo to affordtertiarybutyloxycarbonyl-O-3(methyl)butyl-L-tyrosine N-methyl amide(6.45 g, 17 mM) which crystallised from ethyl acetate/hexane as needlesm.p. 115°-116° C. (Found: C,66.3; H,9.3; N,8.0 C₂₀ H₃₂ N₂ O₄ requires:C,65.9; H,8.9; N,7.7%); δ (CDCl₃) 0.94 (6H,d,J=6 Hz,(CH₃)₂ C); 1.42(9H,s,(CH₃)₃); 1.6-2.0 (3H,m,CH₂ CH) 2.74 (3H,d,J=4 Hz, CH₃ NH); 3.0(2H,m,CH₂ C₆ H₄); 3.9 (2H,t,J=8 Hz,CH₂ O); 4.24 (1H,m,NHCHCO); 5.08(1H,m,NH) 5.8 (1H,m,NH) 6.8, and 7.06 (each 2H,each d,each J=8 Hz,C₆H₄).

EXAMPLE 31

(a)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypropyl]-L-leucyl-O-6(methyl)heptyl-L-tyrosineN-methyl amide

N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-6-methylheptyl-L-tyrosine N-methyl amide (0.16 g, 0.24 mM) in methanol (10 ml)was treated with aqueous sodium hydroxide (0.7 ml, 0.5M) with continuousstirring at 20° C. for 24 h. The reaction was adjusted to pH7 withacetic acid and concentrated in vacuo to afford a white solid which waswashed with ethyl acetate and water and dried in vacuo to yieldN-[3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypropyl]-L-leucyl-O-6(methyl)heptyl-L-tyrosineN-methylamide m.p. 176°-177° C. (Found: C,66.3; H,8.2; N,8.4; C₃₆ H₅₄ N₄O₇ +H₂ O requires: C,64.3; H,8.4; N,8.4%); δ (CDCl₃) 0.84(12H,m,2×(CH₃)₂ C); 1.0-1.8 (14H,m,6×,CH₂,2×(CH); 2.56 (3H,d,J=CH₃ NH);2.4-3.64 (6H,m,2×CH₂,2×CH); 3.88 (2 H,m,CH₂ O); 4.82 (1H,m, NHCHCO); 5.0(2H,s,CH₂ C₆ H₅); 6.74, and 7.06 (each 2H,each d,each J=8 Hz,C₆ H₄);7.18 (1H,m,NH); 7.32 (5H,m,C₆ H₅); 7.8, (1H,m,NH); 6.08 (1H,m,NH).

The methyl ester used in the preceeding section was prepared asdescribed below:

(b)N-[3-N-(Benzoyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-(methyl)heptyl-L-tyrosineN-Methyl amide

N-Tertiarybutyl oxycarbonyl-O-6-methyl heptyl-L-tyrosine-N-methylamide(447 mg, 1.1 mM) in CH₂ Cl₂ (10 ml) was treated with trifluoroaceticacid (10 ml) at 20° C. for 2 h. The solvent was removed in vacuo and theresidue redissolved in ether saturated with hydrogen chloride. This wasrepeated twice to afford O-6-methyl heptyl-L-tyrosine-N-methylamidehydrochloride and treated with N-methyl morpholine (420 mg, 4.4 mM),1-hydroxybenzotriazole (159 mg, 1.04 mM), N-[3-N-(Benzoyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucine hydrochloride (418 mg, 1mM) prepared as described in Example (1) and N-Ethyl-N'-3'dimethylaminopropyl carbodiimide hydrochloride (200 mg, 1.04 mM) at 0° C. Thereaction was adjusted to pH7 with N-methylmorpholine, stirredcontinuously and allowed to warm to 20° C. over 1 h. The reaction wasdiluted with dichloromethane and washed successively with water, aqueoussaturated sodium hydrogen carbonate and aqueous citric acid (1M) driedover sodium sulphate and concentrated in vacuo to afford a gum. Columnchromatography on silica in ethyl acetate/hexane 1:1 affordedN-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-6(methyl)heptyl-L-tyrosine N-methylamide(0.17 g, 0.25 mM) as a colourless oil (Found: [M+H]⁺ =694.213. C₃₇ H₅₆N₄ O₇ requires: [M+H]⁺ =699.4227); δ (CDCl₃) 0.88 (12H,m,2×(CH₃)₂ CH);1.12-1.92 (14H,m,6×CH₂,2×CH) 2.74 (3H,d,J=4 HzCH₃ NH); 2.9-3.42(6H,m,CH₂,2×CH); 3.64 (3H,s,CH₃ O); 3.9 (2H,t,J=5 HzCH₂ NH); 4.6(1H,q,NHCHCO) 5.1 (2H,s,CH₂ C₆ H₅); 5.3 (1H,m,NH); 6.7 (1H,m,NH); 6.76;and 7.06 (each 2H,each d,each J=8 Hz,C₆ H₄); 7.3 (5H,s,C₆ H₅); 7.5(1H,m,NH).

The preceeding intermediate was prepared as described below:

(c) N-Tertiary butyl oxycarbonyl-O-6-methylheptyl-L-tyrosine-N-methylamide

N-Tertiarybutyloxycarbonyl-L-tyrosine (14.1 g, 50 mM) in drydimethylformamide (200 ml) was treated with sodium hydride (3.45 g, 115mM) at 10° C. under an atmosphere of argon for 2 h. 6-Methyl hexylbromide (13.1 g, 50 mM) was added and the reaction stirred at 20° C. for16 h. Water (200 ml) was added and the reaction extracted twice withethyl acetate (400 ml). The aqueous phase was adjusted to pH1 withhydrochloric acid (6M) and extracted twice with ethyl acetate (400 ml)and the ethyl acetate extracts combined, dried over sodium sulphate andconcentrated in vacuo to a gum. Column chromatography on silica in ethylacetate afforded crude N-tertiarybutyloxycarbonyl-L-tyrosine (4.9 g) asa colourless foam which was used directly in the next step. This acid(4.9 g) in dichloromethane (150 ml) was treated with methylaminehydrochloride (0.8 g, 25 mM), 1-hydroxybenzotriazole (2.1 g, 14 mM),dicylclohenylcarbodiimide (2.8 g, 14 mM) adjusted to pH7 withN-methylmorpholine at 0° C. and stirred continuously as the reaction wasallowed to warm to 20° C. over 16 h. The reaction was filtered, washedwith water, aqueous saturated sodium hydrogen carbonate and aqueouscitric acid (1M), dried over sodium sulphate and concentrated in vacuoto a gum. Column chromatography on silica in ethyl acetate/hexane 1:1affordedN-tertiarybutyloxycarbonyl-O-6(methylheptyl)-L-tyrosine-N-methyl amidewhich crystallised from ethyl acetate as needles m.p. 103°-106° C.(Found: C, 67.5; H,9.5; N,.9. C₂₃ H₃₈ N₂ O₄ requires: C,68.0; H,9.4;N,6.9%); δ (CDCl₃) 0.88 (6H,d,J=6 Hz, (CH₃)₂ C); 1.12-1.86 (9H,m,(CH₂)₄CH); 1.4 (9H,s,(CH₃)₃ C); 2.72 (3H,d,J=4 Hz, CH₃ NH); 2.98 (2H,m,CH₂ C₆H₄); 3.9 (2H,t,J=6 Hz,O--CH₂); 4.2 (1H,m,NHCHCO); 5.04 (1H,m,NH); 5.72(1H,m,NH); 6.78, and 7.26 (each 2H,each d,each J=9 Hz,C₆ H₄).

EXAMPLE 32

(a)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypropyl]-L-leucyl-O-benzyl-L-tyrosineN-Methylamide

To a stirred solution of the ester from example (0.36 g) in methanol (10ml) was added dilute sodium hydroxide (1 ml, 1M). After stirring for 48h at room temperature the reaction mixture was neutralised with aceticacid and the product was filtered off. Recrystallisation frommethanol/water gave the title compound as a solid (159 mg); m.p.172°-175°; (Found: C, 65.70; H, 6.91; N, 8.40. C₃₅ H₄₄ N₄ O₇ 0.5H₂ Orequires C, 65.50; H, 7.06; N, 8.73%); δ (d⁶ DMSO) 0.80 (6H, m,CH(CH₃)₂); 1.0-1.35 and 1.44-1.84 (together 5H, NHCH₂ CH₂, CH₂CH(CH₃)₂); 2.4-3.5 (9H, NHCH₂ CH₂ CHCO₂ H, α-CH, CH₂ Tyr, CONHCH₃); 4.43(1H, m, α-CH); 5.02 (2 H, s, CH₂ C₆ H₅); 6.87 (2H, d, J=8 Hz, Tyr); 7.11(2H, d, J=8 Hz, Tyr); 7.2-7.6 (11H, m, C₆ H₅ ×2, CONH); 7.86 (1H, m,CONH); 8.16 (1H, d, J=8 Hz, CONH).

(b)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-benzyl-L-tyrosineN-Methylamide

To a cold (0°) solution ofN-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucine(418 mg, 1 mM) in dichloromethane (50 ml) was added N-methylmorpholine(0.1 g), 1-hydroxybenzotriazole (153 mg, 1 mM) anddicyclohexylcarbodiimide (206 mg, 1 mM). After stirring for 15 minutesat 0° O-benzyl L-tyrosine N-Methylamide hydrochloride (321 mg, 1 mM) andN-methylmorpholine (0.1 g) were added. The reaction mixture afterstirring and warming to room temperature over 4.5 h was filtered andwashed with water. 3N citric acid, saturated aqueous sodium bicarbonatesolution and water. The organic extract was dried and concentrated invacuo to an oil. Column chromatography on silica eluting with 1:1 ethylacetate/hexane in an increasing ethyl acetate gradient then gave thetitle compound as a foam (0.37 g); (Found: [m+1]⁺ =647.3417. C₃₆ H₄₆ N₄O₇ requires 647.3445); δ (CDCl₃) 0.88 (6H, m, CH(CH₃)₂); 1.05-2.2 (6H,m, NHCH₂ CH₂, CH₂ CH(CH₃)₂ ; 2.74 (3H, d, J=5 Hz, CONHCH₃); 2.96-3.44(6H, m, NHCH₂ CH₂ CHCO₂, α-CH, CH₂ Tyr); 3.66 (3H, s, OCH₃); 4.60 (1H,dd, J=7 and 15 Hz, α-CH); 5.02 (2H, s, CH₂ C₆ H₅); 5.09 (2H, s, CH₂ C₆H₅); 6.56 (1H, br, CONH); 6.90 (2H, d, J=8 Hz, Tyr); 7.12 (2H, d, J=8Hz, Tyr); 7.25-7.6 (12H, m, CONH×2, C₆ H₅ ×2).

O-benzyl-L-tyrosine N-Methylamide hydrochloride used in this preparationwas synthesised as follows:

N-tertiarybutoxycarbonyl-O-benzyl-L-tyrosine N-methylamide (3 g) wasadded to a mixture of TFA and CH₂ Cl₂ (1:1 100 ml) at room temperature.After 15 minutes volatiles were removed in vacuo and the residue wasdissolved in water. Neutralisation with solid sodium bicarbonate,extraction into CH₂ Cl₂ and evaporation of the organic extract in vacuothen gave a solid. Co-evaporation of this with ethereal HCl gaveO-benzyl-L-tyrosine N-Methylamide hydrochloride.

(c) N-Tertiarybutoxycarbonyl-O-benzyl-L-tyrosine N-methylamide

To a solution of N-tertiarybutoxycarbonyl-O-benzyl-L-tyrosine (7.4 g, 20mM), 1-hydroxybenzotriazole (3 g, 20 mM), methylamine HCl (1.3 g, 20 mM)and N-methylmorpholine (2 g, 20 mM) in CH₂ Cl₂ (20 ml) stired and cooledto 0° was added DCC (4.2 g, 20 mM). After being allowed to stir and warmto room temperature overnight the reaction mixture was filtered andwashed with saturated aqueous sodium bicarbonate solution, 3N citricacid and brine. The dried organic extract was then concentrated in vacuoto give N-tertiarybutoxycarbonyl-O-benzyl-L-tyrosine N-methylamide as asolid. Recrystallisation from dichloromethane/hexane gave (4.5 g); m.p.165°-172°; (Found: C, 68.85; H, 7.43; N, 7.39. C₂₂ H₂₈ N₂ O₄ requires C,68.73; H, 7.34; N, 7.29%); δ (CDCl₃) 1.40 (9H, s, OC(CH₃)₃); 2.73 (3H,d, J=5 Hz, CONHCH₃); 2.96 (2H, m, CH₂ Tyr); 4.25 (1H, m, α-CH); 5.03(2H, s, CH₂ C₆ H₅); 5.80 (1H, br, CONH); 6.86 (2H, d, J=8 Hz, Tyr); 7.12(2H, d, J=8 Hz, Tyr); 7.3-7.5 (5H, m, C₆ H₅).

EXAMPLE 33

(a)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypropyl]-L-leucyl-O-cyclopentyl-L-tyrosineN-Methylamide

To a solution ofN-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-Leucyl-O-cyclopentyl-L-tyrosineN-Methylamide (536 mg, 0.86 mM) in methanol (10 ml) was added dilutesodium hydroxide (1.8 ml, 1M). After stirring for 24 h at roomtemperature the reaction mixture was filtered, neutralised with aceticacid and concentrated in vacuo to a solid. Purification from ethylacetate/water gave the title compound as a solid (245 mg); m.p.160°-170°; (Found: C, 64.36; H, 7.71; N, 9.18. C₃₃ H₄₆ N₄ O₇ +0.25H₂ Orequires C, 64.42; H, 7.62; N, 9.11); δ (d⁶ DMSO) 0.8 (6H, M, CH(CH₃)₂);1.17 (2H, m, CH₂ CH(C₃)); 1.45-2.0 (11H, m, NHCH₂ CH₂, CH₂ CH(CH₃)₂,CH-alkyl); 2.58 (3H, d, J=5 Hz, CONHCH₃); 2.6-3.65 (6H, M, NHCH₂ CH₂CHCO₂ H, α-CH, CH₂ C₆ H₄); 4.42 (1H, q, J=7 and 15 Hz, α-CH); 4.73 (1H,m, O-CH(cyclopentyl)); 5.02 (2H, s, CH₂ C₆ H₅); 6.77 (2H, d, J=8 Hz, C₆H₄); 7.08 (2H, d, J=8 Hz, C₆ H₄); 7.25 (1H, br, CONH); 7.35 (5H, s, C₆H₅); 7.85 (1H, br, CONH); 8.15 (1H, d, J=Hz, CONH).

The preceeding ester was synthesised as described below:

(b)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-cyclopentyl-L-tyrosineN-Methylamide

To a solution ofN-[3-N-(Benzyoxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucine(418 mg, 1 mM) in dichloromethane (10 ml) was added N-methylmorpholine(106 mg, 1 mM) and the solution was stirred and cooled to 0°.1-hydroxybenzotriazole (161 mg, 1.05 mM),N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (202 mg,1.05 mM) and N-methylmorpholine (106 mg, 1 mM) were then added. After 15minutes a solution of O-cyclopentyl-L-tyrosine N-methylamide (1.1 mM) indichloromethane (5 ml) (which had been prepared by treatment of theN-tertiarybutoxycarbonyl precursor with trifluoroacetic acid andsubsequent neutralisation with N-methylmorpholine) was added. Thereaction after being allowed to stir and warm to room temperatureovernight was diluted with dichloromethane (30 ml), washed with water,saturated aqueous sodium bicarbonate and water. The dried organicextract was concentrated in vacuo to give the title compound as an oil(0.55 g); (Found: [m+1]⁺ =625.3608. C₃₄ H₄₉ N₄ O₇ requires 625.3601); δ(CDCl₃) 0.82 (6H, m, CH(CH₃)₂); 1.0-2.0 (11H, m, CH₂ CH(CH₃)₂,CH-cyclopentyl); 2.7(3H, d, J=Hz, CONHCH₃); 2.9-3.5 (6H, m, NHCH₂ CH₂CHCO₂ H,α-CH, CH2Tyr); 3.65 (3H, s, OCH₃); 4.58 (1H, dd, J=7 and 15 Hz,α-CH); 4.68 (1H, br, ?); 5.08 (2H, s, CH₂ C₆ H₅); 5.22 (1H, br, CONH);6.68 (1H, br, CONH); 6.78 (2H, d, J=8 Hz, Tyr); 7.10 (2H, d, J=8 Hz,Tyr); 7.32 (5H, s, C₆ H₅); 7.52 (1H, d, J=8 Hz, CONH).

The N-(Tertiarybutoxycarbonyl)-O-cyclopentyl-L-tyrosine N-Methylamideused in this preparation was prepared as follows:

(c) N-(Tertiarybutoxycarbonyl)-O-cyclopentyl-L-tyrosine N-Methylamide

To a cold (0°) solution ofN-(Tertiarybutoxycarbonyl)-O-cyclopentyl-L-tyrosine (6.89 g, 20 mM) indichloromethane (40 ml) was added 1-hydroxybenzotriazole (3.32 g, 21 mM)and dicyclohenylcarbodiimide (4.47 g, 21 mM). After 20 minutes at 0° asolution of methylamine (1.34 g, 40 mM) in dichloromethane (20 ml) wasadded and the reaction mixture was allowed to stir and warm to roomtemperature overnight. The reaction mixture after filtration and washingwith water, dilute (1M) sodium hydroxide, 3N citric acid and water wasdried and concentrated in vacuo to an oil. Recrystallisation from ethylacetate/hexane gave the title compound as white crystals (1 g); m.p.152°-154°; (Found: C, 66.77; H, 8.47; N, 7.83. C₂₀ H₃₀ N₂ O₄ requires C,66.27; H, 8.34; N, 7.73%); δ(CDCl₃) 1.40 (9H, s, C(CH₃)₃); 1.5-2.0 (8H,CH-cyclopentyl); 2.72 (3H, d, J=5 Hz, CONHCH₃); 2.95 (2H, m, CH₂ Tyr);4.22 (1H, dd, J=7 and 15 Hz,α-CH); 4.72 (1H, br, O--CH (cyclopentyl));5.07 (1H, br, CONH); 5.75 (1H, br, CONH); 6.78 (2H, d, J=8 Hz, C₆ H₄);7.10 (2H, d, J=8 Hz, C₆ H₄).

The N-(Tertiarybutoxycarbonyl)-O-cyclopentyl-L-tyrosine used in thispreparation was prepared as follows:

(d) N-(Tertiarybutoxycarbonyl)-O-cyclopentyl-L-tyrosine

To a cold (10°) stirred solution of N-tertiarybutoxycarbonyl-L-tyrosine(11.25 g, 40 mM) in dry DMF (150 ml) under argon was added sodiumhydride (2.76 g, 80% dispersion in oil, 92 mM). After one hour at 10°cyclopentyl bromide (5.96 g, 40 mM) was added and the reaction wasallowed to warm and stir to room temperature overnight. Water (200 ml)and dichloromethane (300 ml) were then added and the aqueous layer wasseparated. The separated aqueous layer was washed with dichloromethane,acidified with hydrochloric acid and re-extracted with dichloromethane(300 ml×2). These latter dichloromethane extracts were dried andconcentrated in vacuo to an oil. Column chromatography of this materialon silica eluting with ethyl acetate gave the title compound as a foam(7.05 g). This was used directly in the next step without furtherpurification.

EXAMPLE 34

(a)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypropyl]-L-leucyl-O-(2-aminobutyl-2-oxo)ethyl-L-tyrosineN-Methylamide

To a solution ofN-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-(2-aminobutyl-2-oxo)ethyl-L-tyrosineN-Methylamide (199 mg, 0.3 mM) in methanol (10 ml) was added dilutesodium hydroxide (0.5 ml, 1M). After stirring for 90 h at roomtemperature the reaction mixture was neutralised with acetic acid andconcentrated in vacuo. Purification from ethyl acetate/water gave thetitle compound as a solid (143 mg); m.p. 164°-170°; (Found: C, 59.44; H,7.39; N, 10.49 C₃₄ H₄₉ N₅ O₈ +1.5H₂ O requires C, 59.81; H, 7.68; N,10.26%); δ(d⁶ DMSO) 0.7-1.0 (9H, m, CH₂ CH₃, CH(CH₃)₂); 1.2-2.0 (9H, m,NHCH₂ CH₂, NHCH₂ (CH₂)₂ CH₃, CH₂ CH(CH₃)); 2.62-3.64 (9H, m, NHCH₂ CH₂CHCO₂ H, CH, CH₂ Tyr, CONHCH₃ ; 4.64 (2H, s, OCH₂ CO); 4.56 (1H,m,α-CH); 5.07 (2H, s, CH₂ C₆ H₅); 6.90 (2H, d, J=8 Hz, Tyr); 7.17 (2H,d, J=8 Hz, Tyr); 7.34 (5H, br s, C₆ H₅).

(b)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-(2-aminobutyl-2-oxo)ethyl-L-tyrosineN-Methylamide

To a cold (0°) stirred solution ofN-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucineHCl in CH₂ Cl₂ /DMF (11 ml, 10:1) was added N-methylmorpholine (240 mg,2.4 mM), 1-hydroxybenzotriazole (180 mg, 1.2 mM) andN-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.24 g,1.2 mM). After stirring for 15 min. at 0° a solution of n-butylamine (90mg, 1.2 mM) in CH₂ Cl₂ was added and the reaction mixture was allowed towarm and stir to room temperature overnight. The reaction mixture afterdilution with CH₂ Cl₂ (30 ml) was washed (water (10 ml), 3N citric acid(10 ml) and saturated aqueous sodium bicarbonate (10 ml)), dried,filtered and concentrated in vacuo to an oil. Crystallisation from ethylacetate/hexane then gave the title compound as a white solid (0.27 g);m.p. 101°-107°; (Found: C, 62.13; H, 7.84; N, 10.42. C₃₅ H₅₁ N₅ O₈+0.5H₂ O requires C, 61.93; H,7.72; N, 10.32%); δ(CDCl₃) 0.87 (6H, m,CH₂ CH(CH₃)₂); 0.94 (3H, t, J=7 Hz, CH₂ CH₃); 1.04-2.0 (9H, m,NHCH₂ CH₂,CH₂ CH(CH₃)₂, NHCH₂ (CH₂)₂ CH₃); 2.75 (3H, d, J=5 Hz, CONHCH₃);2.85-3.45 (9H, M, NHCH₂ CH₂ CHCO₂ H, NHCH₂, α-CH, CH₂ Tyr); 3.66 (3H, s,OCH₃); 4.42 (2H, s, OCH₂ CO); 4.63 (1H, dd, J=7 and 15 Hz, α-CH); 5.07(2H, s, CH₂ C₆ H₅); 5.26 (1H, br, CONH); 6.63 (1H, br, CONH); 6.82 (2H,d, J=8 Hz, Tyr); 7.15 (2H, d, J=8 Hz, Tyr); 7.34 (5H, s, C₆ H₅); 7.58(1H, d, J=10 Hz, CONH).

EXAMPLE 35

(a)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypropyl]-L-leucyl-O-(2'-carboxyethyl-L-leucylN-Methylamide)-L-tyrosine N-Methylamide

To a solution ofN-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-(2'-carboxyethyl-L-leucylN-Methylamide)-L-tyrosine N-Methylamide (300 mg, 0.4 mM) in methanol (10ml) was added dilute sodium hydroxide (1.2 ml, 0.5M). After stirring for48 h at room temperature the reaction mixture was neutralised withacetic acid and concentrated in vacuo. Recrystallisation frommethanol/water gave the title compound as a solid (115 mg); m.p.162°-163°; (Found: C, 60.26; H, 7.49; N, 11.09 C₃₇ H₅₄ N₆ O₉ +0.5H₂ Orequires C, 60.39; H, 7.53; N, 11.42%); δ(d⁶ DMSO) 0.7-0.9 (12H, m,CH(CH₃)₂ ×2); 0.95-1.85(8H, m, CH₂ CH(CH₃)×2, NHCH₂ CH₂); 2.45-3.7 (13H,m, NHCH₂ CH₂ CHCO₂ H, CONHCH₃ ×2, α-CH×2, CH₂ Tyr); 4.34 (1H, m, --CH);4.46 (2H, s, OCH₂ CO); 5.05 (2H, s, CH₂ C₆ H₅); 6.82 (2H, d, J=8 Hz,Tyr); 7.13 (2H, d, J=8 Hz, Tyr); 7.24 (1H, br, CONH); 7.35 (5H, s, C₆H₅); 7.85 (1H, br, CONH); 7.98 (2H, br, CONH×2); 8.15 (1H, d, J=10 Hz,CONH).

The ester required in this preparation was prepared as follows:

(b)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-(2'-carboxyethyl-L-leucylN-Methylamide)-L-tyrosine N-Methylamide

To a cold (0°) solution ofN-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-(2'-carboxyethyl)-L-tyrosineN-Methylamide (0.6 g, 1.1 mM) in CH₂ Cl₂ /DMF (11 ml 10:1) was added1-hydroxybenzotriazole (0.18 g, 1.2 mM),N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.24 g,1.2 mM) and N-methylmorpholine (0.24 g, 2.4 mM). After 5 min. at 0°L-leucine N-methylamide HCl (0.22 g, 1.2 mM) and N-methylmorpholine (120mg, 1.2 mM) were added and the reaction was allowed to stir and warm toroom temperature overnight. The reaction mixture after diluting with CH₂Cl₂ (30 ml) was washed with water (20 ml), 3N citric acid (30 ml) andsaturated aqueous sodium bicarbonate solution. The organic extract wasdried and concentrated in vacuo to an oil. Recrystallisation from ethylacetate/hexane gave the title compound as crystals (350 mg); m.p.122°-123°; (Found: C,61.16; H, 7.45; N,11.14. C₃₈ H₅₆ N₆ O₉ requiresC,61.60; H,7.62; N,11.34%); δ(CDCl₃) 0.8-1.0 (12H, m, CH(CH₃)₂ ×2);1.05-2.05 (8H,m, CH₂ CH(CH₃)₂ ×2, NHCH₂ CH₂); 2.75 (3H, d, J=5 Hz,CONHCH₃); 2.80 (3H, d, J=5 Hz, CONHCH₃); 2.8-3.5 (7H, m,NHCH₂ CH₂ CHCO₂,CH₂ Tyr, α-CH×2); 3.68 (3H, s, OCH₃); 4.44 (2H, s,OCH₂ CO); 4.4-4.7 (2H,m,α-CH×2); 5.10 (2H, s, CH₂ C₆ H₅); 5.34 (1H, t,J=6 Hz, CONH); 6.42 (1H,br, CONH); 6.73 (1H, br, CONH); 6.82 (2H, d, J=8 Hz, Tyr); 7.0 (1H, br,CONH); 7.15 (2H, d, J=8 Hz, Tyr), 7.36 (5H, s, C₆ H₅); 7.56 (1H, br,CONH).

EXAMPLE 36

(a)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypropyl]-L-leucyl-O-methyl-L-tyrosineN-Phenethylamide

To a solution ofN-[3-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosineN-Phenethylamide (210 mg, 0.33 mM) in methanol (5 ml) was added dilutesodium hydroxide (0.5 ml, 1M). After stirring for 5 h at roomtemperature the reaction mixture was concentrated in vacuo and dilutedwith water. The aqueous extract after washing with ether was acidifiedto pH 3 with dilute (1N) HCl. The resulting solid was filtered and driedto give the title compound as a solid (110 mg); (Found: C, 65.5; H,7.0;N, 9.1. 0.05H₂ O requires C, 65.5; H, 7.0; N, 8.7%).

(b)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosineN-Phenethylamide

To a cold (0°) solution ofN-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosineHydrochloride (400 mg, 0.67 mM) in DMF was added N-methylmorpholine (68mg, 0.67 mM), 1-hydroxybenzotriazole (107 mg, 0.7 mM) and benzylamine(72 mg, 0.67 mM). The reaction mixture was then cooled to -10° andN-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (134 mg,0.7 mM) and N-methylmorpholine (71 mg, 0.7 mM) were added. After beingallowed to warm and stir to room temperature over 20 h the reactionmixture was concentrated in vacuo. The resultant residue afterdissolving in CH₂ Cl₂ and washing (water, saturated aqueous sodiumbicarbonate solution) was concentrated in vacuo. Recrystallisation fromethyl acetate/hexane gave the title compound (290 mg); (Found: C, 66.9;H, 7.1; N, 8.7. required C, 67.2; H, 7.3; N, 8.6%); δ(CDCl₃) 0.86 (6H,M, CH(CH₃)₂); 1.1-1.9 (6H, m, NHCH₂ CH₂, CH₂ CH(CH₃)₂); 2.95-3.42 (6H,m, NHCH₂ CH₂ CHCO₂ CH₃,α-CH, CH₂ Tyr); 3.45 (3H, s, OCH₃); 3.73 (3H, s,OCH₃); 4.38 (2H, m, CH₂ C₆ H₅); 4.65 (1H, q, J=7 and 15 Hz, α-CH); 5.04(1H, br, CONH); 5.08 (2H, s, CH₂ C₆ H₅); 6.78 (2H, d, J=8 Hz, Tyr); 6.96(1H, br, CONH); 7.11 (2H, d, J=8 Hz, Tyr); 7.10-7.30 (5H, m, C₆ H₅);7.34 (5H, s, C₆ H₅); 7.54 (1H, d, J=8 Hz, CONH).

The starting material for this reaction was prepared as follows:

(c)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosineN-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosine t-butyl ester (3 g) wasdissolved in TFA (30 ml) and water (1.5 ml). After 4 h at roomtemperature volatiles were removed in vacuo and the residue wasco-evaporated twice with toluene. Trituation of the resulting solid withethereal HCl then gaveN-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosineas its hydrochloride salt.

(d)N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosine^(t) Butyl ester

A solution of methyl 4-N-(benzyloxycarbonyl)amino-2-bromo-butanoate (2.5g, 7.5 mM), L-leucyl-O-methyl-L-tyrosine t-butyl ester (1.82 g, 5 mM),(which had been prepared by hydrogenolysis of the N-benzloxycarbonylprecursor in the usual manner), and N-methylmorpholine (0.76 g, 7.5 mM)in acetonitrile (15 ml) was heated under reflux for 30 h. The reactionmixture was then concentrated in vacuo, dissolved in CH₂ Cl₂, washed(water, 3N citric acid and saturated aqueous sodium bicarbonatesolution), dried and evaporated in vacuo to yield an oil. Columnchromatography on silica eluting with 40:60 ethyl acetate/hexane gavethe title compound as an oil (670 mg); δ(CDCl₃) 0.88 (6H, dd, J=7 Hz,CH(CH₃)₂); 1.42 (9H, s, OC(CH₃)₃); 1.62 (3H, m, CH₂ CH(CH₃)₂); 1.86 (2H,m, NHCH₂ CH₂); 2.9-3.36 (6H, m, NHCH₂ CH₂ CHCO₂ CH₃,α-CH, CH₂ Tyr); 3.66(3H, s, OCH₃); 3.78 (3H, s, OCH₃); 4.68 (1H, dd, J=7 and 15 Hz,α-CH);5.09 (2H, s, CH₂ C₆ H₅); 5.16 (1H, br, CONH); 6.80 (2H, d, J=8 Hz, Tyr);7.01 (1H, br, CONH); 7.07 (2H, d, J=8 HZ, Tyr); 7.35 (5H, s, C₆ H₅).

The N-(benzyloxycarbonyl)-L-leucyl-O-methyl-L-tyrosine t-butyl esterused in this preparation was prepared as follows:

(e) N-(Benzyloxycarbonyl)-L-leucyl-O-methyl-L-tyrosine ^(t) Butyl ester

To a cold (0°) solution of N-tertiarybutoxycarbonyl-L-leucine (4 g, 15.1mM), O-methyl-L-tyrosine t-butyl ester (3.8 g, 15.1 mM) and1-hydroxybenzotriazole (2.43 g, 15.9 mM) in DMF (30 ml) was addedN-methylmorpholine (1.61 g, 15.9 mM) andN-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.04 g,15.9 mM). After stirring at 0° for 1 h the solution was allowed to warmto room temperature overnight. The reaction mixture was thenconcentrated in vacuo, dissolved in ethyl acetate, washed (water, 0.5mole citric acid, brine, saturated aqueous sodium bicarbonate solutionand brine), dried and evaporated in vacuo to give an oil. Chromatographyon silica eluting with 25% ethyl acetate in hexane then gave the titlecompound as a foam (4.65 g); (Found: [m+1]⁺ =499.2803. C₂₈ H₃₉ N₂ O₆requires 499.2808); δ(CDCl₃) 0.92 (6H, m, CH(CH₃)₃); 1.42 (9H, s,OC(CH₃)₃); 1.4-1.83 (3H, m, CH₂ CH(CH₃)₂); 3.03 (2H, m, CH₂ Tyr); 3.76(3H, s, OCH₃); 4.18 (1H, m,α-CH); 4.69 (1H, dd, J=7 and 15 Hz,α-CH);5.12 (2H, s, CH₂ C₆ H₅); 5.17 (1H, m, CONH); 6.43 (1H, m, CONH); 6.79(2H, d, J=8 Hz, Tyr); 7.06 (2H, d, J=8 Hz, Tyr); 7.36 (5H, s, C₆ H₅).

The O-methyl-L-tyrosine t-butyl ester used in this synthesis wasprepared as follows:

(f) O-methyl-L-tyrosine t-butyl ester

To a cold, (-5°), stirred solution ofN-benzyloxycarbonyl-O-methyl-L-tyrosine (6.58 g, 20 mM) int-butanol/pyridine (70 ml, 5:2) was added phosphorus oxychloride (3.35g, 22 mM) dropwise over 0.5 h. After a further 0.5 h at -5° the reactionwas warmed to room temperature and was then left to stir overnight. Thereaction mixture, after diluting with ethyl acetate and washing (0.5Mcitric acid, brine, saturated aqueous sodium bicarbonate solution andbrine) was concentrated in vacuo to give the required t-butyl ester(7.54 g). To a portion of this (7.4 g, 19.2 mM) in methanol (100 ml) wasadded 10% Pd/C (1 g) and acetic acid (1.16 g, 19.2 mM). After stirringthe reaction mixture under hydrogen at room temperature for 4 h it wasfiltered and evaporated in vacuo. The residue was then taken up indichlromethane and saturated aqueous sodium bicarbonate solution and theorganic phase was separated, washed with brine, dried and evaporated invacuo to yield O-methyl-L-tyrosine t-butyl ester as a solid (3.95 g).

The compounds of Examples 37 to 143 and their routes of preparation areexemplified within the following Tables.

Using the methods illustrated in examples 1-24 further examples 37-143in Table 1 are prepared.

CompoundsN-[2-(S)-N-(1-(R)-carboxyethyl)amino-4,4-di-(trifluoromethyl)butanoyl]-O-methyl-L-tyrosineN-methylamide andN-[2-(S)-N-(3-(benzyloxycarbonyl)amino-1-(R)-carboxypropyl)amino-4,4-di-(trifluoromethyl)butanoyl]-O-methyl-L-tyrosineN-methylamide are likewise prepared by methods described in examples1-24.

    TABLE 1               STERO- MP. °C..sup.2 MP. °C..sup.2 No PROCESS A     A.sup.1 Y n R.sup.2 R.sup.3 A.sup.3 CHEM.sup.1 R.sup.1 = OCH.sub.3     R.sup.1 =      OH                                                               37 1A     -- H -- 1 H CH(CH.sub.3)CH.sub.2 CH.sub.3 GlyNHC.sub.4 H.sub.9 n RS     82-84 74-77 38 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 GlyNHC.sub.4     H.sub.9 n RS  87-95 39 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2     GlyNHC.sub.4 H.sub.9 n SS  175-180 40 1A -- H -- 1 H CH.sub.2      CH(CH.sub.3).sub.2 ValNHC.sub.6 H.sub.13 n RSS  190-193 41 1A -- H -- 1     H CH.sub.2 CH(CH.sub.3).sub.2 ValNHC.sub.6 H.sub.13 n SSS  200-203 42 1A     -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 LeuNHC.sub.4 H.sub.9 n RSS     138-139 180-185 43 1A -- H -- 1 H CH.sub.2      CH(CH.sub.3).sub.2 LeuNHC.sub.4 H.sub.9 n SSS 180-185 183-185 44 1A --     H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 LeuNHC.sub.4 H.sub.9 n RSR 103-107     150-160 45 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 LeuNHC.sub.4     H.sub.9 n SSR 94-98 185-188 46 1A -- H -- 1 H CH.sub.2      CH(CH.sub.3).sub.2 Thr(OBZ)NHC.sub.4 H.sub.9 n RSSR 62-67 145-148 47 1A     -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHC.sub.4 H.sub.9 n SSSR     61-64 147-152 48 1A -- H -- 3 H CH.sub.2      CH(CH.sub.3).sub.2 ValGlyOCH.sub.3 RS  87-92 49 1A -- H -- 3 H CH.sub.2     CH(CH.sub.3).sub.2 ValGlyOCH.sub.3 SS  177-180 50 1A -- H -- 1 H     CH.sub.2 CH(CH.sub.3).sub.2 ValGlyOCH.sub.3 RS 51 1A -- H -- 1 H     CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3 RSSR 72-76 194-197 52 1A     -- H -- 1 H CH.sub.2      CH(CH.sub.3).sub.2 Thr(OBZ)NH(CH.sub.2).sub.2SCH.sub.2 CH.sub.3 RSSR     .sup. 162-164.sup.3 105-109 53 1A -- H -- 1 H CH.sub.2      CH(CH.sub.3).sub.2 Thr(OBZ)NH(CH.sub.2).sub.2SOCH.sub.2 CH.sub.3 RSSR     80-85 54 1A -- H -- 1 H CH.sub.2      CH(CH.sub.3).sub.2 Thr(OBZ)NH(CH.sub.2).sub.3CONH.sub.2 RSSR 97 193 55     1A -- H -- 1 H CH.sub.2      CH(CH.sub.3).sub.2 Thr(OBZ)NH(CH.sub.2).sub.5CONH.sub.2 RSSR foam     115-120 56 1A -- H -- 1 H CH.sub.2                                       C      CH(CH.sub.3).sub.2 Thr(OBZ)N(CH.sub.3).sub.4 H.sub.9 n RSSR oil 56-57     57 1A -- H -- 1 H CH.sub.2      CH(CH.sub.3).sub.2 Thr(OBZ)NH(CH.sub.2).sub.2SO.sub.2 CH.sub.2 CH.sub.3     RSSR .sup. 157-161.sup.3 90-95 58 1A -- H -- 1 H CH.sub.2      CH(CH.sub.3).sub.2 Thr(OBZ)NH(CH.sub.2).sub.5CO.sub.2 H RSSR .sup.     131-133.sup.3 105-107 59 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2     NHCH(CONHCH.sub.3)CH.sub.2NHCO.sub.2 C(CH.sub.3).sub.3 RSS 107-112     174-182 60 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Ser(OBZ)NHCH.sub.3     RSS 61-64 188-190 61 1A -- H -- 1 H CH.sub.2      CH(CH.sub.3).sub.2 TyrNHCH.sub.3 RSS  212-217 62 1A -- H -- 1 H     CH.sub.2 CH(CH.sub.3).sub.2 NHCH(CONHCH.sub.3)CH.sub.2NHCOPh RSS 71-74     186-191 63 1A -- H -- 1 H CH.sub.2      CH(CH.sub.3).sub.2 NHCH(CONHCH.sub.3).sub.2NHZ RSS 110-112 163-166 64     1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 ThrNH(CH.sub.2).sub.3     CONH.sub.2 RSSR  235 65 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2     AlaNHC.sub.4 H.sub.9 n RSS .sup. 174-176.sup.3 158-162 66 1A -- Z NH 2 H     CH.sub.2 CH(CH.sub.3).sub.2 AlaNHCH.sub.3 SSS  176-182 67 1A -- Z NH 2 H     CH.sub.2 CH(CH.sub.3).sub.2 AlaNHCH.sub.3 RSS  166-168 68 1A -- H -- 6 H     CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3 SSSR 62-64 112-120 69 1A     -- H -- 6 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3 RSSR 79-80     63-66 70 1A -- Z NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3     RSSR 92-94 160-164 71 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2     AlaNHCH.sub.3 RSS 87-90 84-88 72 1A -- H -- 1 H CH.sub.2      CH(CH.sub.3).sub.2 PheNHCH.sub.3 RSS 116-119 115-116 73 1A -- H -- 1 H     CH.sub.2 CH(CH.sub.3).sub.2 SarNHCH.sub.3 RS 160-175 77-80 74 1A -- H --     1 H CH.sub.2 CH(CH.sub.3).sub.2 ProNHCH.sub.3 RSS  99-102 100-105 75 1A     -- H -- 1 H CH.sub.2      CH(CH.sub.3).sub.2 NHCH(CONHCH.sub.3)(CH.sub.2).sub.6 CH.sub.3 RS(RS)     .sup. 155-159.sup.3 186-191 76 1A -- H -- 1 H CH.sub.2      CH(CH.sub.3).sub.2 NCH.sub.3Tyr(OBZ)NHCH.sub.3 RSS .sup. 115-120.sup.3     115-121 77 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 iso-AbaNHCH.sub.3:     RS .sup. 150-153.sup.3 177-179 78 1A -- H -- 1 H CH.sub.2      CH(CH.sub.3).sub.2 NZLysNHCH.sub.3 RSS .sup. 170-172.sup.3 162-164 79     1A Z Leu NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3     SRSS  145-150 80 1A -- H -- 1 H CH.sub.2      CH(CH.sub.3).sub.2 SerNHCH.sub.3 RSS  191-198 81 1A -- Z NH 2 H     CH.sub.2 CH(CH.sub.3).sub.2 AlaNHCH.sub.3 SSS  176-182 82 1A -- Z NH 2 H     CH.sub.2 CH(CH.sub.3).sub.2 AlaNHCH.sub.3 RSS  166-168 83 1B -- H NH 1 H     CH.sub. 2 CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3 RSSR  105-107 84 1B --     CH.sub.3 CO NH 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3 RSSR     131-132 108-112 85 1B -- CH.sub.3 CO NH 1 H CH.sub.2 CH(CH.sub.3).sub.2     Thr(OBZ)NHCH.sub.3 RRSR 133-134 100-102 86 1B -- (CH.sub.3).sub.3 COCO     NH 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3 RSSR 95-97 114-118     87 1B -- (CH.sub.3).sub.3 COCO NH 1 H CH.sub.2 CH(CH.sub.3).sub.2     Thr(OBZ)NHCH.sub.3 RSSR 123-124 80-90 88 1B DnpPro Leu NH 1 H CH.sub.2     CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3 SSRSSR  112-115 89 1B DnpPro Leu     NH 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3 SSRRSR  100-115 90     1B -- Ph(CH.sub.2).sub.2 NHCO -- 1 H CH.sub.2 CH(CH.sub.3).sub.2     Tyr(OCH.sub.3)NHCH.sub.3 R(RS)S 124-128 91 1B -- HO.sub.2 C -- 1 H     CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 64-66 130-132     92 2A --  H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OBZ)NH.sub.2 RSS     .sup. 117-119.sup.4 193-196 93 2A -- H -- 1 H CH.sub.2      CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS .sup. 96-97.sup.4     200-202 94 2A -- H -- 1 H CH(CH.sub.3).sub.2 AlaNHC.sub.4 H.sub.9 n SSS     60-62 100-105 95 2A -- H -- 1 H CH(CH.sub.3).sub.2 AlaNHC.sub.4 H.sub.9     n RSS  165-170 96 2A -- H -- 1 H CH.sub.3 AlaNHC.sub.4 H.sub.9 n (RS)SS     220-223 97 2A -- H -- 1 H CH.sub.3 AlaNHC.sub.4 H.sub.9 n (SR)SS     231-234 98 2A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 His(BZ)NHCH.sub.3     RSS  95-103 99 2A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 AlaNHOCH.sub.3     RSS  192-196 100 2A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OC.sub.4     H.sub.9 t)NHCH.sub.3 RSSR   98-108 101 2A -- H -- 1 H CH.sub.2      CH(CH.sub.3).sub.2 TyrNH.sub.2 RSS  219-230 102 2A -- H -- 1 H (CH.sub.2     ).sub.2 CH.sub.3 AlaNHC.sub.4 H.sub.9 n R(RS)S 84-85 199-201 103 2A --     CH.sub.3 CO NH 3 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3     RSS 101-104 150 104 2A -- H -- 1 H (CH.sub.2).sub.2      SCH.sub.3 AlaNHC.sub.4 H.sub.9 n RSS foam 155-159 105 2A -- H -- 1 H     CH.sub.2 CH(VH.sub.3).sub.2 NHCH(CH.sub.3)CH.sub.2 Ph RS(RS) 106 2A -- H     -- 1 H CH.sub.2 C.sub.6 H.sub.5 AlaNHC.sub.4 H.sub.9 n (RS)SS  173-178     107 2A -- H -- 1 H CH.sub.2 OCH.sub.2 Ph AlaNHC.sub.4 H.sub.9 n RSS     158-162 108 2A -- H -- 1 H CH(CH.sub.3)CH.sub.2 CH.sub.3 AlaNHC.sub.4     H.sub.9 n RSS 90-94 162-164 109 2A -- Ph(CH.sub.2).sub.2 CO NH 2 H     CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 114-118 162-163 1     10 2A -- H -- 1 (CH.sub.2).sub.5 AlaNHC.sub.4 H.sub.9 n (RS)S  95-105     111 2A -- (CH.sub.3).sub.2 CHCH.sub.2 CO NH 2 H CH.sub.2      CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS  168-172 112 2A --     CH.sub.3 OCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3     RSS  149-153 113 2A Z Pro NH 2 H CH.sub.2      CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 SRSS 102-103 174-179 114 2A     -- (CH.sub.3).sub.2 CHCH.sub.2 OCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2     Tyr(OCH.sub.3)NHCH.sub.3 RSS  171-175 115 2A -- PhCHCHCO NH 2 H CH.sub.2     CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS  188-191 116 2A --     2-ClC.sub.6 H.sub.4 CO NH 2 H CH.sub.2      CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 109 158-163 117 2A --     4-ClC.sub.6 H.sub.4 CO NH 2 H CH.sub.2      CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 105-108 187-192 118 2A     -- Z NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)OC.sub.4 H.sub.9 t     RSS  101-102 119 2A -- 4-CH.sub.3C.sub.6 H.sub.4 CH.sub.2 NH 2 H     CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 141-143 164-167        OCO 120 2A -- 4-ClC.sub.6 H.sub.4 CH.sub.2 OCO NH 2 H CH.sub.2     CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS  167-171 121 2A --     HO.sub.2 C(CH.sub.2).sub.2 CO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2     Tyr(OCH.sub.3)NHCH.sub.3 RSS 50-55 160-171 122 2A -- 4-CH.sub.3C.sub.6     H.sub.4 CO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3     RSS 98 190-194 123 2A -- PhCH.sub.2 CO NH 2 H CH.sub.2 CH(CH.sub.3).sub.     2 Tyr(OCH.sub.3)NHCH.sub.3 RSS  173-179 124 2A -- 2-ClC.sub.6 H.sub.4     CH.sub.2 OCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3     RSS  168-174 125 2A -- 4-CH.sub.3 OC.sub.6 H.sub.4 CH.sub.2 NH 2 H     CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS  162-166    OCO     126 2A -- Bornyl-OCO NH 2 H CH.sub.2      CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS  145-150 127 2A --     2-CH.sub.3C.sub.6 H.sub.4 CH.sub.2 NH 2 H CH.sub.2 CH(CH.sub.3).sub.2     Tyr(OCH.sub.3)NHCH.sub.3 RSS  170-173    OCO 128 2A -- Ph(CH.sub.2).sub.2      OCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS     147-151 129 2A -- PhCH.sub.2 SO.sub.2 NH 2 H CH.sub.2 CH(CH.sub.3).sub.2     Tyr(OCH.sub.3)NHCH.sub.3 RSS 50-60 174-178 130 2A -- PhCH.sub.2      N(CH.sub.3)CO NH 2 H CH.sub.2      CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 65-70 90-95 131 2A --     2-NaphthylCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3     RSS 148-153 162-172 132 2A -- 1-NaphthylCO NH 2 H CH.sub.2      CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 67-71 167-173 133 2A --     Ph -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS     161-166 134 2A -- 1-NaphthylCH.sub.2OCO NH 2 H CH.sub.2      CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 85-86 182-184 135 2A --     2-NaphthylCH.sub.2OCO NH 2 H CH.sub.2      CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 92-98 168-171 136 2A --     PhCCCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS     137 2A -- H -- 1 H CH.sub.2 CH(CF.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3     RSS 138 2A -- Z NH 2 H CH.sub.2      CH(CF.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 139 2A -- Z NH 2 H     CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OBZ)NHCH.sub.3 RSS  172-175 140 2A -- Z     NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OC.sub.5 H.sub.11 n)NHCH.sub.3     RSS 81-85 155-157 141 2A ZPro Leu NH 2 H CH.sub.2 CH(CH.sub.3).sub.2     Tyr(OCH.sub.3)NHCH.sub.3 SSRSS 142 2A ZPro Pro NH 2 H CH.sub.2      CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 SSRSS 143 2B -- HO.sub.2 C     -- 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 133-135     110-120     Notes for TABLE 1:     .sup.1 Stereochemistryoptical centres labelled from left to right.     .sup.2 Of hydrated form where appropriate.     .sup.3 m.p. of HCl salt.     .sup.4 R.sup.1 = OC.sub.2 H.sub.5 not OCH.sub.3   Gly = glycyl =     NHCH.sub.2 CO     ##STR22##     ##STR23##     Ph = phenyl = C.sub.6 H.sub.5     Bz = CH.sub.2 C.sub.6 H.sub.5     Z =  PhCH.sub.2 O.CO     DMP = 2,4dinitrophenyl     ##STR24##     Sar = Sarcosyl = N(CH.sub.3)CH.sub.2 CO     ##STR25##     ##STR26##     ##STR27##     ##STR28##     ##STR29##     ##STR30##

The activities of representative compounds according to the inventionare given below in Table II.

                  TABLE II                                                        ______________________________________                                                                                     7                                             IC.sub.50 (μM)                                                             Human Rheumatoid                                                 Example No.  Synovial Collagenase                                             ______________________________________                                         5           1.7                                                               6           42                                                                7           5.5                                                               9           9.5                                                              11           0.8                                                              13           91                                                               14           1.2                                                              15           3.1                                                              16           4.9                                                              18           1.3                                                              19           51                                                               21           11                                                               22           42                                                               23           25                                                               24           19                                                               25           1.2                                                              26            0.74                                                            27           2.1                                                              28           3.6                                                              29            0.54                                                            30           1.1                                                              31           2.5                                                              32            0.91                                                            33            0.74                                                            34           2.6                                                              35           2.4                                                              36           1.0                                                              ______________________________________                                    

One of the preferred compounds in accordance with the present inventionwas tested in vivo to demonstrate the antiarthritic activity thereof ina standardized animal model test and compared against prednisolone whichhas been shown to be clinically effective in the treatment of rheumatoidarthritis.

The therapeutic effectivness is assessed in the rat model ofinflammatory arthritis described by Trentham et al. (1977). In thismodel, the arthritis, induced by an intradermal injection of Type IIcollagen, generally develops in both hind paws and the progression andseverity of the lesion are monitored by physical measurements of themalleolar width and ankle extensibility.

The compound of Example II in the present specification was administeredby intraperitoneal injections at 100 mg/kg twice daily for 21 dayscommencing seven days after the injection of the disease.

The results of the foregoing test are set forth in Table III below.

TABLE III

The effect ofN-[3-N-(benzyloxycarbonyl)amino-1-(R)-carboxypropyl]-L-leucyl-O-methyl-L-tyrosine-N-methylamide(Example 11) on the Physical Parameters of Arthritic Rats' Paws

    ______________________________________                                                        PERCENTAGE CHANGE                                                             FROM CONTROL                                                                  EXPERIMENT                                                                    NUMBER     MEAN                                               PARAMETER         1      2       3   x                                        ______________________________________                                        MALLEOLAR WIDTH                                                               Example 11        65     43      41  5.0.                                     Prednisolone      98     96      97  97                                       ANKLE EXTENSION                                                               Example 11        66     39      39  48                                       Prednisolone      1.0..0.                                                                              96      95  97                                       ______________________________________                                    

Drugs (Example 11 or prednisolone) and vehicle were administered togroups of 10 rats each. Physical measurements on the rat paws were madeweekly for four weeks and consisted of (a) the distance between themedial and lateral malleolus (malleolar width) and (b) the ankleextension which was measured by applying a standard force to th rat pawand measuring the angle formed between the flexor surface of the footand the tibia. Evidence of a therapeutic effect was denoted by asignificant reduction in malleolar width and a significant increase inankle extension. Both measurements were carried out in apparatusdesigned for the purpose. Area-under-the-curve data from these timecourse experiments was examinaed by non-parametic statistical analysis.The prednisolone standard was given at a dose of 0.625 mg/kg b.i.d.Prednisolone was chosen as a standard since steroids are known to beeffective both clinically in Rheumatoid Arthritis and in animal modelsof arthritis.

References

Trentham, D. E., Townes, A. S. & Kang, A. H. (1977) "Autoimmunity toType II Collagen: An Experimental Model of Arthritis" J. Expt. Med. 146.857-868.

What is claimed is:
 1. A compound of the formula: ##STR31## and thepharmaceutically acceptable acid addition salts thereof wherein R'₂represents hydroxy, alkoxy, cycloalkoxy, aralkoxy or substituted alkoxywherein the substituent is selected from alkylaminocarbonyl or the group##STR32## R'₃ represents alkylamino or aralkylamino; and thestereochemistry of the carbon atom marked by the asterisk is R or S or adiastereomeric mixture thereof.
 2. A compound according to claim 1wherein R'₂ is straight or branched chain alkoxy of from about 1 to 6carbon atoms.
 3. A compound according to claim 2 wherein R'₂ is selectedfrom n-propoxy or n-pentoxy.
 4. A compound according to claim 1 whereinR'₃ is alkylamino.
 5. A compound according to claim 4 wherein saidalkylamino is methylamino.
 6. A compound according to claim 1 whereinthe stereochemistry of the carbon atom marked by the asterisk is R.
 7. Acompound according to claim 1 of the formula. ##STR33## or apharmaceutically acceptable acid addition salt thereof.
 8. A compoundaccording to claim 1 of the formula ##STR34## or a pharmaceuticallyacceptable acid addition salt thereof.
 9. A method of promoting anantiarthritic effect in a mammal in need thereof comprisingadministering thereto a collagenase inhibiting effective amount of acompound according to claim
 1. 10. A method according to claim 9 whereinsaid compound is of the formula: ##STR35##